Heterothallic sexual reproduction in three canker-inducing tree pathogens within the <i>Fusarium torreyae</i> species complex

<i>Fusarium zanthoxyli</i> and <i>F. continuum</i> are sister taxa that are the etiological agents of canker disease of prickly ash (<i>Zanthoxylum bungeanum</i>) in northern China. These two pathogens, together with <i>F. torreyae</i>, the causal agent of canker disease of the critically endangered conifer Florida torreya (<i>Torreya taxifolia</i>) from northern Florida and southwestern Georgia, constitute a novel clade, the <i>F. torreyae</i> species complex. To assess their reproductive mode, a polymerase chain reaction (PCR) assay targeting the <i>MAT1-1</i> and <i>MAT1-2</i> idiomorphs was designed and validated, using <i>MAT</i> sequences mined from the whole-genome sequence of the three <i>F. torreyae</i> clade pathogens and several closely related fusaria. Results of the <i>MAT</i> idiomorph PCR assay indicated that isolates of the three pathogens were <i>MAT1-1</i> or <i>MAT1-2</i>. When <i>MAT1-1</i> and <i>MAT1-2</i> isolates of each species were crossed on carrot agar, all of the <i>F. zanthoxyli</i> (N = 30) and <i>F. continuum</i> (N = 3) isolates tested were female fertile, yielding mature perithecia with viable ascospores. By comparison, only one pairing of the five different isolates of <i>F. torreyae</i> produced perithecia; however, the majority of the asci in this cross aborted or produced fewer than eight ascospores. Of the three temperatures tested (i.e., 22, 25, and 27 C), the optimal temperature for perithecium production was 22–25 C in <i>F. zanthoxyli</i> and 25 C in <i>F. continuum</i> and <i>F. torreyae</i>. Ascospore progeny from three separate crosses of <i>F. zanthoxyli</i> and <i>F. continuum</i> and one cross of <i>F. torreyae</i> were genotyped to assess whether they were the products of genetic recombination and sexual reproduction. Genotyping of 34–40 progeny from the <i>F. zanthoxyli</i> and <i>F. continuum</i> crosses confirmed that they were the products of sexual reproduction. However, only 36% of the progeny in the <i>F. torreyae</i> cross were recombinant, which was roughly half of the nonparental progeny expected with three markers segregating.