Continuous passaging in vitro can lead to the accumulation of changes in DNA sequence that potentially affect the properties of microbes, making them different from the original isolates. The identification of such genetic alterations is rare in fungi. A set of insertional mutants in the plant pathogenic fungus Leptosphaeria maculans, all derived from the same transformation experiment, had independent Agrobacterium T-DNA insertions and reduced pathogenicity on canola (Brassica napus). None of the insertions co-segregated in progeny from crosses with the reduction in pathogenicity. Genome sequences of three strains were analysed, and a mutation identified in a gene (ptf1, for pathogenicity-associated transcription factor 1) encoding a putative Zn₂(II)Cys₆ transcription factor. Homologs are found in other ascomycetes, and are required for pathogenicity by Fusarium graminearum, Fusarium oxysporum and Magnaporthe oryzae. The mutation in the L. maculans ptf1 gene co-segregates in progeny from crosses with the reduction in pathogenicity, a strain with an independent mutant allele isolated using CRISPR-Cas9 editing has reduced pathogenicity, and addition of wild type copies of the gene restores pathogenicity. Thus, this work defines a base pair substitution that occurred during in vitro passaging of a fungus that contributed to an attenuation of pathogenicity.

体外连续传代会导致 DNA 序列变化的积累，这些变化可能会影响微生物的特性，使它们与原始分离株不同。这种遗传改变的鉴定在真菌中很少见。植物病原真菌Leptosphaeria maculans中的一组插入突变体，均来自相同的转化实验，具有独立的农杆菌T-DNA 插入，并降低了对油菜 ( Brassica napus ) 的致病性。随着致病性的降低，没有一个插入在后代中与杂交共分离。分析了三个菌株的基因组序列，并在一个基因 ( ptf1，对于致病性相关转录因子 1) 编码推定的 Zn ₂ (II)Cys ₆转录因子。在其他子囊菌中发现了同源物，并且是禾谷镰刀菌、尖孢镰刀菌和稻瘟病菌的致病性所必需的。L. maculans ptf1基因的突变在后代中与致病性降低的杂交共分离，具有使用 CRISPR-Cas9 编辑分离的独立突变等位基因的菌株降低了致病性，添加野生型基因拷贝恢复了致病性. 因此，这项工作定义了在体外发生的碱基对替换 导致致病性减弱的真菌的传代。