Phenolic degradation by catechol dioxygenases is associated with pathogenic fungi with a necrotrophic lifestyle in the Ceratocystidaceae

DATA AVAILABILITY : The datasets used and/or analyzed in this study are available in the NCBI Database (https://www.ncbi.nlm.nih.gov/nucleotide/). Accession numbers are detailed in Table 1.Fungal species of the Ceratocystidaceae grow on their host plants using a variety of different lifestyles, from saprophytic to highly pathogenic. Although many genomes of fungi in the Ceratocystidaceae are publicly available, it is not known how the genes that encode catechol dioxygenases (CDOs), enzymes involved in the degradation of phenolic plant defense compounds, differ among members of the Ceratocystidaceae. The aim of this study was therefore to identify and characterize the genes encoding CDOs in the genomes of Ceratocystidaceae representatives. We found that genes encoding CDOs are more abundant in pathogenic necrotrophic species of the Ceratocystidaceae and less abundant in saprophytic species. The loss of the CDO genes and the associated 3-oxoadipate catabolic pathway appears to have occurred in a lineage-specific manner. Taken together, this study revealed a positive association between CDO gene copy number and fungal lifestyle in Ceratocystidaceae representatives.The National Research Foundation and the Max Planck Society.http://www.g3journal.orgam2023BiochemistryForestry and Agricultural Biotechnology Institute (FABI)GeneticsMicrobiology and Plant PathologyZoology and Entomolog

Agrobacterium-mediated transformation of Ceratocystis albifundus

Functional association between genomic loci and specific biological traits remains lacking in many fungi, including the African tree pathogen Ceratocystis albifundus. This is mainly because of the absence of suitable transformation systems for allowing genetic manipulation of this and other fungi. Here, we present an optimized protocol for Agrobacterium tumefaciens-mediated transformation of C. albifundus. Strain AGL-1 of A. tumefaciens and four binary T-DNA vectors (conferring hygromycin B or geneticin resistance and/or expressing the green fluorescent protein [GFP]) were used for transforming germinated conidia of three isolates of C. albifundus. Stable expression of these T-DNA-encoded traits was confirmed through sequential sub-culturing of fungal transformants on selective and non-selective media and by using PCR and sequence analysis. Single-copy integration of the respective T-DNAs into the genomes of these fungi was confirmed using Southern hybridization analysis. The range of experimental parameters determined and optimised included: (i) concentrations of hygromycin B and geneticin required for inhibiting growth of the wild type fungus and (ii) the dependence of transformation on acetosyringone for inducing the bacterium’s virulence genes, as well as (iii) the duration of fungus-bacterium co-cultivation periods and (iv) the concentrations of fungal conidia and bacterial cells used for the latter. The system developed in this study is stable with a high-efficiency, yielding up to 400 transformants per 106 conidia. This is the first report of a transformation protocol for C. albifundus and its availability will be invaluable for functional studies in this important fungus.Supplementary Figure S1. Restriction map of the plasmids used in this study (A: pBHt-2; B: pCAMBIA0380; C: pC-g-418-GFP; D: pC-HYG-GFP). HygR= hygromycin B resistance cassettes, ZsGreen 1= gfp gene, g-418= geneticin resistance cassette and KanR= kanamycin resistance cassette.Supplementary Figure S2. Effect of different parameters (number of conidia and concentration of bacterial cells) during co-cultivation at 25 °C with A. tumefaciens used for ATMT on Ceratocystis albifundus strains CMW 13980 (A) CMW 4068 (B) and CMW40625 (C) for the 4 vectors included in this study (pC-HYG-GFP, pC-g-418-GFP, pCAMBIA0380 and pBHt2). Bars represent standard error, where different letters indicate significantly different means (p < 0.05) based on Tukey’s tests. The letters a-c show differences among bacterial cell concentrations (optical density at 600nm) relative to specific conidial concentrations, while the letters x-z, show differences among conidial concentration relative to specific bacterial cell concentrations.Supplementary Figure S3. A, B: Agarose gel of PCR products obtained with hph specific primers from the C. albifundus transformants carrying plasmids pC-HYG-GFP (A), pBHt2 (A) and pCAMBIA0380 (B). A: Randomly selected hygromycin-resistant transformants obtained with the binary vectors pC-HYG-GFP (lanes 2-3: CMW 13980; lanes 5-6: CMW 4068 and lanes 9-10: CMW 40625) and pBHt-2 (lanes 4: CMW 13980; 7-8: CMW 4068; 11-12: CMW 40625) were used as PCR templates. Lane 13: plasmid DNA of pBHt-2 used as positive control; lane 14: negative control; lane M: 100bp DNA ladder (Invitrogen, USA). B: gDNA of randomly selected hygromycin resistant transformants carrying the binary vector pCAMBIA0380 (lanes 2–3: CMW 13980; 4-5: CMW 4068; 6-7: CMW 40625) were used as PCR templates. Lane 8: plasmid DNA of pCAMBIA0380 used as positive control; lane M: 100bp DNA ladder (Invitrogen, USA). C, D: Agarose gel of PCR products obtained with gfp specific primers from the C. albifundus transformants carrying plasmids pC-HYG-GFP (A), pBHt-2 (A) and pC-g-418-GFP (B). C: gDNA of randomly selected gfp-expressed transformants obtained with the binary vectors pC-HYG-GFP (lanes 1-2: CMW 13980; lanes 4-5: CMW 4068 and lanes 8-9: CMW 40625) and pBHt-2 (lanes 3: CMW 13980; 6-7: CMW 4068; 10-11: CMW 40625) were used as PCR templates. Lane 12: plasmid DNA of pBHt-2 used as positive control; lane 13: negative control; lane M: 100bp DNA ladder (Invitrogen, USA). D: gDNA of randomly selected hygromycin resistant transformants carrying the binary vector pC-g-418-GFP (lanes 2–3: CMW 13980; 4-5: CMW 4068; 6-7: CMW 40625) were used as PCR templates. Lane 8: plasmid DNA of pC-g-418-GFP used as positive control; lane 9: negative control; lane M: 100bp DNA ladder (Invitrogen, USA). E. Agarose gel of PCR products obtained with g-418 specific primers from the C. albifundus transformants. gDNA of randomly selected geneticin resistant transformants obtained with the binary vectors pC-g-418-GFP (lanes 1–3: 13980; 4-7: CMW 4068; 8-11: CMW 40625) were used as PCR templates. The positive control, plasmid DNA of pC-g-418-GFP (lane 12). The negative control, gDNA of C. albifundus wild type (lane 13). Lane M contains 100bp DNA ladder (Invitrogen, USA).Supplementary Figure S4. Fluorescence micrographs of C. albifundus transformants showing constitutive expression of GFP. A-H. CMW 13980, I-P. CMW4068 and Q-X. CMW 40625. A, C, E: Bright field image of the CMW 13980 hyphae carrying plasmids pC-HYG-GFP, pC-g-418-GFP and pBHt2, respectively; B, D, F: Green fluorescence of the same hyphae. I, K, M: Bright field image of the CMW 4068 hyphae carrying plasmids pC-HYG-GFP, pC-g-418-GFP and pBHt2, respectively; J, L, N: Green fluorescence of the same hyphae. Q, S, U: Bright field image of the CMW 40625 hyphae carrying plasmids pC-HYG-GFP, pC-g-418-GFP and pBHt2, respectively. R, T, V: Green fluorescence of the same hyphae G, O, W: Bright field image of the wild type control of strains CMW 13980, CMW 4068 and CMW 40625, respectively, H, P and X: Same hyphae under the fluorescent microscope.Supplementary Figure S5. Colony diameter of C. albifundus isolates CMW 13980, CMW 4068 and CMW 40625 after 21 d at 25 °C on 2% MEA amended with different concentrations of either kanamycin (A) or carbenicillin (B).The South African Department of Science and Technology (DST) and National Research Foundation (NRF).http://www.elsevier.com/locate/micres2020-09-01hj2020BiochemistryForestry and Agricultural Biotechnology Institute (FABI)GeneticsMicrobiology and Plant Patholog

Copy number variation discovery in South African Nguni-sired and Bonsmara-sired crossbred cattle

SUPPLEMENTARY MATERIALS : TABLE S1: Information on the crossbred animals used in this study, TABLE S2: Sequencing depth and mapped reads of Nguni-sired and Bonsmara-sired crossbreds, FIGURE S1: CNV summary statistics of Copy number (CN) and number of CNVs for each CN class, FIGURE S2: Bar plot displaying the contribution of each copy number class to the total number of CNV calls per chromosome for all crossbred individuals, TABLE S3: All the CNVs detected in the crossbreds.DATA AVAILABILITY STATEMENT : The raw data supporting the conclusions of this article will be made available by the authors upon reasonable request in line with ARC intellectual property regulations.Crossbreeding forms part of Climate-Smart beef production and is one of the strategies to mitigate the effects of climate change. Two Nguni-sired and three Bonsmara-sired crossbred animals underwent whole genome sequencing. Following quality control and file preparation, the sequence data were investigated for genome-wide copy number variation (CNV) using the panelcn.MOPS tool. A total of 355 CNVs were identified in the crossbreds, of which 274 were unique in Bonsmara-sired crossbreds and 81 unique in the Nguni-sired crossbreds. Genes that differed in copy number in both crossbreds included genes related to growth (SCRN2, LOC109572916) and fertility-related factors (RPS28, LOC1098562432, LOC109570037). Genes that were present only in the Bonsmara-sired crossbreds included genes relating to lipid metabolism (MAF1), olfaction (LOC109569114), body size (HES7), immunity (LOC10957335, LOC109877039) and disease (DMBT1). Genes that were present only in the Nguni-sired crossbreds included genes relating to ketosis (HMBOX1) and amino acid transport (LOC109572916). Results of this study indicate that Nguni and Bonsmara cattle can be utilized in crossbreeding programs as they may enhance the presence of economically important traits associated with both breeds. This will produce crossbred animals that are good meat producers, grow faster, have high fertility, strong immunity and a better chance of producing in South Africa’s harsh climate conditions. Ultimately, this study provides new genetic insights into the adaptability of Nguni and Bonsmara crossbred cattle.Climate change plays a major role in livestock production. Hence the utilization of crossbreeding strategies allows for the improvement of animal production during harsh environmental conditions. The aim of this study was to identify the genetic differences in the F1 Nguni × Bonsmara and its reciprocal cross (Bonsmara × Nguni). This was achieved by studying the changes in structural variation, such as copy number variants in these two crosses. The major findings from this study have revealed several genes relating to adaption in these crossbred cattle.The National Research Foundation of South Africa.https://www.mdpi.com/journal/animalsBiochemistryGenetic

Unisexual reproduction in Huntiella moniliformis

Sexual reproduction in fungi is controlled by genes present at the mating type (MAT) locus, which typically harbors transcription factors that influence the expression of many sex-related genes. The MAT locus exists as two alternative idiomorphs in ascomycetous fungi and sexual reproduction is initiated when genes from both idiomorphs are expressed. Thus, the gene content of this locus determines whether a fungus is heterothallic (self-sterile) or homothallic (self-fertile). Recently, a unique sub-class of homothallism has been described in fungi, where individuals possessing a single MAT idiomorph can reproduce sexually in the absence of a partner. Using various mycological, molecular and bioinformatic techniques, we investigated the sexual strategies and characterized the MAT loci in two tree wound-infecting fungi, Huntiella moniliformis and Huntiella omanensis. H. omanensis was shown to exhibit a typically heterothallic sexual reproductive cycle, with isolates possessing either the MAT1-1 or MAT1-2 idiomorph. This was in contrast to the homothallism via unisexual reproduction that was shown in H. moniliformis, where only the MAT1-2-1 gene was present in sexually reproducing cultures. While the evolutionary benefit and mechanisms underpinning a unisexual mating strategy remain unknown, it could have evolved to minimize the costs, while retaining the benefits, of normal sexual reproduction.University of Pretoria, the Department of Science and Technology (DST)/National Research Foundation (NRF) Centre of Excellence in Tree Health Biotechnology and the Genomics Research Institute (University of Pretoria Institutional Research Theme) and the National Research Foundation of South Africa. specific unique reference number (UID) 83924).http://www.elsevier.com/locate/yfgbi2016-07-31hb201

QTL mapping of mycelial growth and aggressiveness to distinct hosts in Ceratocystis pathogens

Some species of Ceratocystis display strong host specificity, such as C. fimbriata sensu stricto that is restricted to sweet potato (Ipomoea batatas) as host. In contrast, the closely related C. manginecans, infects Acacia mangium and Mangifera indica but is not pathogenic to I. batatas. Despite the economic importance of these fungi, knowledge regarding the genetic factors that influence their pathogenicity and host specificity is limited. A recent inheritance study, based on an interspecific cross between C. fimbriata and C. manginecans and the resultant 70 F1 progeny, confirmed that traits such as mycelial growth rate, spore production and aggressiveness on A. mangium and I. batatas are regulated by multiple genes. In the present study, a quantitative trait locus (QTL) analysis was performed to determine the genomic loci associated with these traits. All 70 progeny isolates were genotyped with SNP markers and a linkage map was constructed. The map contained 467 SNPs, distributed across nine linkage groups, with a total length of 1203 cm. Using the progeny genotypes and phenotypes, one QTL was identified on the linkage map for mycelial growth rate, one for aggressiveness to A. mangium and two for aggressiveness to I. batatas (P < 0.05). Two candidate genes, likely associated with mycelial growth rate, were identified in the QTL region. The three QTLs associated with aggressiveness to different hosts contained candidate genes involved in protein processing, detoxification and regions with effector genes and high transposable element density. The results provide a foundation for studies considering the function of genes regulating various quantitative traits in Ceratocystis.Members of the Tree Protection Cooperative Programme, based at the Forestry and Agricultural Biotechnology Institute, and the Genomics Research Institute at the University of Pretoria, as well as the National Research Foundation, South Africa (Grant UID: 89619 and 95875) and the DST/NRF SARChI chair in Fungal Genomics.http://www.elsevier.com/locate/yfgbi2020-10-01hj2020BiochemistryForestry and Agricultural Biotechnology Institute (FABI)GeneticsMicrobiology and Plant Patholog

Ceratocystidaceae exhibit high levels of recombination at the mating-type (MAT) locus

Mating is central to many fungal life cycles and is controlled by genes at the mating-type (MAT) locus. These genes determine whether the fungus will be self-sterile (heterothallic) or self-fertile (homothallic). Species in the ascomycete family Ceratocystidaceae have different mating strategies, making them interesting to consider with regards to their MAT loci. The aim of this study was to compare the composition of the MAT locus flanking regions in 11 species of Ceratocystidaceae representing four genera. Genome assemblies for each species were examined to identify the MAT locus and determine the structure of the flanking regions. Large contigs containing the MAT locus were then functionally annotated and analysed for the presence of transposable elements. Genes typically flanking the MAT locus in sordariomycetes were found to be highly conserved in the Ceratocystidaceae. The different genera in the Ceratocystidaceae displayed little synteny outside of the immediate MAT locus flanking genes. Even though species ofCeratocystis did not show much synteny outside of the immediate MAT locus flanking genes, species of Huntiella and Endoconidiophora were comparatively syntenic. Due to the high number of transposable elements present in Ceratocystis MAT flanking regions, we hypothesise that Ceratocystis species may have undergone recombination in this region.The National Research Foundation (NRF), members of the Tree Protection Co-Operative Programme (TPCP), the Department of Science and Technology (DST)/NRF Centre of Excellence in Tree Health Biotechnology (CTHB) and SARChI Chair in Fungal Genomics, South Africa.http://www.elsevier.com/locate/funbio2019-12-01hj2019BiochemistryForestry and Agricultural Biotechnology Institute (FABI)GeneticsMicrobiology and Plant Patholog

Homothallism : an umbrella term for describing diverse sexual behaviours

Sexual reproduction is notoriously complex in fungi with species able to produce sexual progeny by utilizing a variety of different mechanisms. This is even more so for species employing multiple sexual strategies, which is a surprisingly common occurrence. While heterothallism is relatively well understood in terms of its physiological and molecular underpinnings, homothallism remains greatly understudied. This can be attributed to it involving numerous genetically distinct mechanisms that all result in self-fertility; including primary homothallism, pseudohomothallism, mating type switching, and unisexual reproduction. This review highlights the need to classify these homothallic mechanisms based on their molecular determinants and illustrates what is currently known about the multifaceted behaviours associated with homothallism.The University of Pretoria, the Department of Science and Technology (DST)/National Research Foundation (NRF) Centre of Excellence in Tree Health Biotechnology and the Genomics Research Institute (University of Pretoria Institutional Research Theme) as well grants from the National Research Foundation of South Africa (including Grant specific unique reference number (UID) 83924).http://www.imafungus.orgam201

Mutualism and asexual reproduction influence recognition genes in a fungal symbiont

Mutualism between microbes and insects is common and alignment of the reproductive interests of microbial symbionts with this lifestyle typically involves clonal reproduction and vertical transmission by insect partners. Here the Amylostereum funguseSirex woodwasp mutualism was used to consider whether their prolonged association and predominance of asexuality have affected the mating system of the fungal partner. Nucleotide information for the pheromone receptor gene rab1, as well as the translation elongation factor 1a gene and ribosomal RNA internal transcribed spacer region were utilized. The identification of rab1 alleles in Amylostereum chailletii and Amylostereum areolatum populations revealed that this gene is more polymorphic than the other two regions, although the diversity of all three regions was lower than what has been observed in free-living Agaricomycetes. Our data suggest that suppressed recombination might be implicated in the diversification of rab1, while no evidence of balancing selection was detected. We also detected positive selection at only two codons, suggesting that purifying selection is important for the evolution of rab1. The symbiotic relationship with their insect partners has therefore influenced the diversity of this gene and influenced the manner in which selection drives and maintains this diversity in A. areolatum and A. chailletii.The National Research Foundation (NRF), members of the Tree Pathology Cooperative Programme (TPCP) and the THRIP initiative of the Department of Trade and Industry (DTI), South Africa.http://www.elsevier.com/locate/funbiohb201

Unexpected placement of the MAT1-1-2 gene in the MAT1-2 idiomorph of Thielaviopsis

Sexual reproduction in the Ascomycota is controlled by genes encoded at the mating-type or MAT1 locus. The two allelic versions of this locus in heterothallic species, referred to as idiomorphs, are defined by the MAT1-1-1 (for the MAT1-1 idiomorph) and MAT1-2-1 (for the MAT1-2 idiomorph) genes. Both idiomorphs can contain additional genes, although the contents of each is typically specific to and conserved within particular Pezizomycotina lineages. Using full genome sequences, complemented with conventional PCR and Sanger sequencing, we compared the mating-type idiomorphs in heterothallic species of Thielaviopsis (Ceratocystidaceae). The analyses showed that the MAT1-1 idiomorph of T. punctulata, T. paradoxa, T. euricoi, T. ethacetica and T. musarum harboured only the expected MAT1-1-1 gene. In contrast, the MAT1-2 idiomorph of T. punctulata, T. paradoxa and T. euricoi encoded the MAT1-2-1, MAT1-2-7 and MAT1-1-2 genes. Of these, MAT1-2-1 and MAT1-2-7 are genes previously reported in this idiomorph, while MAT1-1-2 is known only in the MAT1-1 idiomorph. Phylogenetic analysis showed that the Thielaviopsis MAT1-1-2 groups with the known homologues of this gene in other Microascales, thus confirming its annotation. Previous work suggests that MAT1-1-2 is involved in fruiting body development, a role that would be unaffected by its idiomorphic position. This notion is supported by our findings for the MAT1 locus structure in Thielaviopsis species. This also serves as the first example of a MAT1-1-specific gene restricted to only the MAT1-2 idiomorph.The Claude Leon Foundation in the form of Postdoctoral Fellowship to PMW, the University of Pretoria and the Department of Science and Technology (DST)- National Research Foundation (NRF) Centre of Excellence in Tree Health Biotechnology and the SARCHI chair in Fungal Genomics.http://www.elsevier.com/locate/yfgbi2019-04-01hj2018BiochemistryForestry and Agricultural Biotechnology Institute (FABI)GeneticsMicrobiology and Plant Patholog

Characterization of host-specific genes from pine- and grass-associated species of the Fusarium fujikuroi species complex

SUPPLEMENTARY MATERIALS : FIGURE S1. Phylogenetic relationship of paralogs with regards to the respective host-range-associated gene. FCIR = Fusarium circinatum; FFRAC = Fusarium fracticaudum; FPIN = Fusarium pininemorale; FSUB = Fusarium subglutinans; FIGURE S2. Host-range-associated genes with ancestral origins that emerged within the FFSC. The investigated host-range-associated genes are highlighted in yellow; FCIR = Fusarium circinatum and FTEMP = Fusarium temperatum; FIGURE S3. Host-range-associated genes with ancestral origins that emerged within the FFSC and FOSC. The investigated host-range-associated genes are highlighted in yellow; FCIR = Fusarium circinatum and FTEMP = Fusarium temperatum; FIGURE S4. Host-range-associated genes with ancestral origins that emerged within the broader Fusarium outside the FFSC and FOSC. The investigated host-range associated genes are highlighted in yellow; FCIR = Fusarium circinatum and FTEMP = Fusarium temperatum; FIGURE S5. Host-range-associated genes with less than 10 ancestral origin hits and mostly Fusarium. The investigated host-range-associated genes are highlighted in yellow; FCIR = Fusarium circinatum and FTEMP = Fusarium temperatum; FIGURE S6. Host-range-associated genes with ancestral origins hits and mostly not Fusarium. The investigated host-range-associated genes are highlighted in yellow; FCIR = Fusarium circinatum and FTEMP = Fusarium temperatum; FIGURE S7. Host-range-associated genes with ancestral origins outside Fusarium but in the Ascomycetes. The investigated host-range-associated genes are highlighted in yellow; FCIR = Fusarium circinatum and FTEMP = Fusarium temperatum; FIGURE S8. Host-range-associated genes with ancestral origins outside Fungi. The investigated host-range-associated genes are highlighted in yellow; FCIR = Fusarium circinatum and FTEMP = Fusarium temperatum; FIGURE S9. The distribution of host-range-associated genes from pine-host-associated Fusarium species and conservation of synteny across and between chromosomes and genomes. Pine-host-associated genes distribution across each of the chromosomes as indicated by the blue lines. The conservation of synteny and inversion between the relevant genomes are indicated in the brown blocks and red lines. FCIR = F. circinatum; chromosome size is given in kbp; FIGURE S10. The distribution of hostrange- associated genes from Poaceae-host-associated Fusarium species and conservation of synteny across and between chromosomes and genomes. Poaceae-host-associated gene distribution across each of the chromosomes as indicated by the blue lines. The conservation of synteny and inversion between the relevant genomes are indicated in the brown blocks and red lines. FTEMP = F. temperatum; chromosome size is given in kbp; FIGURE S11. The syntenous relationship between genes from F. circinatum versus F. temperatum; FIGURE S12. The syntenous relationship between genes from F. temperatum and F. circinatum; TABLE S1. BUSCO results for the relevant Fusarium genomes; TABLE S2. The size difference between the chromosomes of four of the six Fusarium species; TABLE S3. Presence of telomeres at chromosomal ends for the two representative Fusarium species examined; TABLE S4. The Blast2GO data for the 72 unique pine-host-associated genes specifically for (A) Fusarium circinatum, (B) F. fracticaudum and (C) F. pininemorale; TABLE S5. The Blast2GO data for the 47 unique Poaceae-host-associated genes, specifically for (A) Fusarium konzum, (B) F. subglutinans and (C) F. temperatum; TABLE S6. The Fischer exact test data for (A) the 72 unique pine-host-associated genes and (B) the 47 unique Poaceae-host-associated genes; TABLE S7. The EST and RNA-seq data for F. circinatum, obtained from Wingfield et al. [52] and Phasha et al. [53], respectively; TABLE S8. The placement of host-range-associated genes in groups that infer their evolutionary origins; TABLE S9. The gene information for the unique F. circinatum genes, in terms of chromosome location, subtelomeric placement, ancestral origin and BLAST description; TABLE S10. The gene information for the unique F. temperatum genes, in terms of chromosome location, subtelomeric placement, ancestral origin and BLAST description; TABLE S11. The host-range-associated gene density for both F. circinatum and F. temperatum; TABLE S12. The SynChro data for genes downstream and upstream of the host-rangeassociated genes of both the pine- and Poaceae-host-associated Fusarium species; FCIR = Fusarium circinatum and FTEMP = Fusarium temperatum.The Fusarium fujikuroi species complex (FFSC) includes socioeconomically important pathogens that cause disease for numerous crops and synthesize a variety of secondary metabolites that can contaminate feedstocks and food. Here, we used comparative genomics to elucidate processes underlying the ability of pine-associated and grass-associated FFSC species to colonize tissues of their respective plant hosts. We characterized the identity, possible functions, evolutionary origins, and chromosomal positions of the host-range-associated genes encoded by the two groups of fungi. The 72 and 47 genes identified as unique to the respective genome groups were potentially involved in diverse processes, ranging from transcription, regulation, and substrate transport through to virulence/pathogenicity. Most genes arose early during the evolution of Fusarium/FFSC and were only subsequently retained in some lineages, while some had origins outside Fusarium. Although differences in the densities of these genes were especially noticeable on the conditionally dispensable chromosome of F. temperatum (representing the grass-associates) and F. circinatum (representing the pine-associates), the host-range-associated genes tended to be located towards the subtelomeric regions of chromosomes. Taken together, these results demonstrate that multiple mechanisms drive the emergence of genes in the grass- and pine-associated FFSC taxa examined. It also highlighted the diversity of the molecular processes potentially underlying niche-specificity in these and other Fusarium species.The South African Department of Science and Innovation’s South African Research Chair Initiative, the DSI-NRF Centre of Excellence in Plant Health Biotechnology at the Forestry and Agricultural Biotechnology Institute (FABI), University of Pretoria, the Food Safety National Program at the United States Department of Agriculture, Agricultural Research Service.https://www.mdpi.com/journal/pathogensam2023BiochemistryForestry and Agricultural Biotechnology Institute (FABI)GeneticsMicrobiology and Plant Patholog