Identification and characterization of a QTL for growth of Fusarium circinatum on pine-based medium

SUPPLEMENTARY FILE S1: HPLC and GC-MS results showing the broad overview and primary metabolites identified in the pine-based media. SUPPLEMENTARY FILE S2: The primer sequences and PCR protocols used to amplify gene regions in this study. SUPPLEMENTARY FILE S3: Reference mapping of F. circinatum Illumina and MinIon raw reads mapped to the genomes of F circinatum KS17 and F. temperatum. SUPPLEMENTARY FILE S4: Genic information of the identified genes and indel region in the QTL region of F. circinatum. InterProScan and gene ontology information are provided for all genes in this region. Further information on the retrotransposons and repeats that are characteristic of the indel within the QTL region is provided.Fusarium circinatum is an economically important pathogen of pine and resides in the Fusarium fujikuroi species complex. Here we investigated the molecular processes underlying growth in F. circinatum by exploring the association between growth and the nutritional environment provided by the pine host. For this purpose, we subjected a mapping population consisting of F. circinatum X F. temperatum hybrid progeny to an analysis of growth rate on a pine-tissue derived medium. These data, together with the available genetic linkage map for F. circinatum, were then used to identify Quantitative Trait Loci (QTLs) associated with growth. The single significant QTL identified was then characterized using the available genome sequences for the hybrid progeny’s parental isolates. This revealed that the QTL localized to two non-homologous regions in the F. circinatum and F. temperatum genomes. For one of these, the F. circinatum parent contained a two-gene deletion relative to the F. temperatum parent. For the other region, the two parental isolates encoded different protein products. Analysis of repeats, G+C content, and repeat-induced point (RIP) mutations further suggested a retrotransposon origin for the two-gene deletion in F. circinatum. Nevertheless, subsequent genome and PCR-based analyses showed that both regions were similarly polymorphic within a collection of diverse F. circinatum. However, we observed no clear correlation between the respective polymorphism patterns and growth rate in culture. These findings support the notion that growth is a complex multilocus trait and raise the possibility that the identified QTL contains multiple small-effect QTLs, of which some might be dependent on the genetic backgrounds. This study improved our current knowledge of the genetic determinants of vegetative growth in F. circinatum and provided an important foundation for determining the genes and processes underpinning its ability to colonize its host environment.The South African Department of Science and Innovation’s South African Research Chair Initiative and the DSI-NRF Centre of Excellence in Plant Health Biotechnology at the Forestry and Agricultural Biotechnology Institute (FABI), University of Pretoria.https://www.mdpi.com/journal/jofam2023BiochemistryForestry and Agricultural Biotechnology Institute (FABI)GeneticsMicrobiology and Plant PathologyZoology and Entomolog

Draft genome sequence of Annulohypoxylon stygium, Aspergillus mulundensis, Berkeleyomyces basicola (syn. Thielaviopsis basicola), Ceratocystis smalleyi, two Cercospora beticola strains, Coleophoma cylindrospora, Fusarium fracticaudum, Phialophora cf. hyalina, and Morchella septimelata

Draft genomes of the species Annulohypoxylon stygium, Aspergillus mulundensis, Berkeleyomyces basicola (syn. Thielaviopsis basicola), Ceratocystis smalleyi, two Cercospora beticola strains, Coleophoma cylindrospora, Fusarium fracticaudum, Phialophora cf. hyalina and Morchella septimelata are presented. Both mating types (MAT1-1 and MAT1-2) of Cercospora beticola are included. Two strains of Coleophoma cylindrospora that produce sulfated homotyrosine echinocandin variants, FR209602, FR220897 and FR220899 are presented. The sequencing of Aspergillus mulundensis, Coleophoma cylindrospora and Phialophora cf. hyalina has enabled mapping of the gene clusters encoding the chemical diversity from the echinocandin pathways, providing data that reveals the complexity of secondary metabolism in these different species. Overall these genomes provide a valuable resource for understanding the molecular processes underlying pathogenicity (in some cases), biology and toxin production of these economically important fungi

Ras2 is important for growth and pathogenicity in Fusarium circinatum

In this study, we investigated to possible role of Ras2 in Fusarium circinatum- a fungus that causes pine pitch canker disease on many different pine species and has a wide geographic distribution. This protein is encoded by the RAS2 gene and has been shown to control growth and pathogenicity in a number of fungi in a mitogen-activated protein kinase- and/or cyclic adenosyl monophosphate pathway-dependent manner. The aim was therefore to characterize the phenotypes of RAS2 gene knockout and complementation mutants of F. circinatum. These mutants were generated by transforming protoplasts of the fungus with suitable split-marker constructs. The mutant strains, together with the wild type strain, were used in growth studies as well as pathogenicity assays on Pinus patula seedlings. Results showed that the knockout mutant strain produced significantly smaller lesions compared to the complementation mutant and wild type strains. Growth studies also showed significantly smaller colonies and delayed conidial germination in the knockout mutant strain compared to the complement mutant and wild type strains. Interestingly, the knockout mutant strain produced more macroconidia than the wild type strain. Collectively, these results showed that Ras2 plays an important role in both growth and pathogenicity of F. circinatum. Future studies will seek to determine the pathway(s) through which Ras2 controls these traits in F. circinatum.Supplementary data 1. Gene knockout and transformation protocol for Fusarium circinatum using split-marker constructs.Supplementary data 2. Maximum likelihood phylogeny for the RAS2 gene from selected Fusarium and closely related fungal species. Bootstrap values above 60% are shown at the nodes, and the scale bar shows substitutions per site. The tree was rooted using Aspergillus nidulans and Alternaria burnsii as outgroup species. GenBank genome assembly numbers are shown in brackets.The Tree Protection Cooperative Programme (TPCP), the University of Pretoria, and the National Research Foundation (NRF) and Department of Science and Innovation (DSI) via their DSI-NRF Centre of Excellence in Plant Health Biotechnology (CPHB) and the DSI-NRF South African Research Chairs Initiative (SARChI) Chair in Fungal Genomics.http://www.elsevier.com/locate/yfgbi2022-02-24hj2021BiochemistryForestry and Agricultural Biotechnology Institute (FABI)GeneticsMicrobiology and Plant Patholog