Dual-Track Genome Evolution in Curvularia muehlenbeckiae Suggests Host Jump to Pecan via Putative Mini-Chromosome Acquisition and Zn ₂ Cys ₆ -Centric Co-adaptation

Curvularia muehlenbeckiae (P-6) is an emerging fungal pathogens responsible for severe leaf spot disease in pecan (Carya illinoinensis), however, the genomic mechanisms underlying its host jump remain elusive. Here, we present the first near complete genome assembly of P-6 (33.77 Mb), revealing a karyotype of 14 chromosomes, including two putative mini-chromosomes (Chr13/Chr14) that harbor 17% and 22% of the pathogen’s candidate virulence factors, respectively. Notably, Chr14 contains a transposase-flanked secondary metabolite biosynthetic gene cluster (SM_BGC), a configuration often associated with horizontal gene transfer in fungi. Pan-genome analysis exposed a conserved Curvularia virulome (99.55% of P-6 virulence orthogroups) alongside lineage-specific expansions of Major Facilitator Superfamily (MFS) transporters (299 genes) and Zn 2 Cys 6 transcription factors (126 genes) that facilitate adaptation to woody hosts - a signature distinct from graminaceous-infecting Curvularia species. Time-resolved transcriptomics revealed a Zn 2 Cys 6 -centric biphasic infection strategy: an early phase (0.5 h post-inoculation [hpi]) governed by Zn 2 Cys 6 hubs regulating MFS transporters and reactive oxygen species (ROS) detoxification genes, and a late necrotrophic phase (72 hpi) mediated by distinct Zn 2 Cys 6 factors inducing carbohydrate metabolism (AMY1, INV2) and toxin production (PKS7, NRPS3). Weighted Gene Co-expression Network Analysis (WGCNA) confirmed stage-specific modules associated with these Zn 2 Cys 6 transcription factors. Ecological profiling indicated optimal growth at 28°C and pH 5.0–6.0, consistent with subtropical disease epidemiology. Our findings support a dual-track evolutionary model where putative mini-chromosomes may facilitate virulence gene acquisition, while correlated expansions of Zn 2 Cys 6 transcription factors and MFS transporters forms a co-regulated network associated with a biphasic infection strategy, identification of these hubs will provide promising targets for eco-friendly management of pecan leaf spot.