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Pokkah boeng disease (PBD) caused by Fusarium complex is one of the major diseases in sugarcane with great impact on the sugarcane industry worldwide. F. sacchari is the prevalent species responsible for PBD in Guangxi. Albeit of a long history of study on the disease, the mechanisms underlying the pathogenicity of the pathogen are still far from clear. To tackle this challenge, we generated an insertional mutant library by transformation of the conidial spores from F. sacchari strain FF001 via Agrobacterium tumefaciens-mediated transformation (ATMT). A total of 3018 hygromycin B-resistant transformants were obtained. Among 12 transformants randomly selected, nine out of 12 transformants carried a single copy of T-DNA, as verified by PCR and Southern blot analysis. The mutants were further screened for virulence variation on detached leaves and 30 mutants with obvious changes in virulence were obtained, including 21 mutants with significantly attenuated and 9 mutants with enhanced virulence. The sites of insertion were determined by thermal asymmetric interlaced PCR (TAIL-PCR) and sequence alignment to the genome sequence of F. sacchari. The majority of the insertions were found in coding regions or promotor regions, with a few in terminator or intergenic regions. It was observed that a few insertions were accompanied with genome fragment deletion, resulting in more than one genes being disrupted in a single insertion. Among the 22 mutated genes with annotated functions were those encoding alpha-mannosidase, neutral amino acid permease, oligomeric Golgi complex component 4, oligopeptide transporter 2, acyl-CoA dehydrogenase, acetoacetyl-CoA synthetase, carbonic anhydrase, Bud 10 protein, kinesin-related protein bimC, zinc finger protein ASD 4, transcription initiation factor TFIID, cutinase G-box binding protein, acriflavine sensitivity control protein ACR-2, nuclear VCP-like protein, heat shock protein 30, thioredoxin, type 2C protein phosphatase (PP2C), exoribonuclease, selenoprotein, DNA polymerase eta, survival factor 1, and aldehyde dehydrogenase, several of which, such as zinc finger protein ASD4 in Magnaporthe oryzae, cutinase G-box binding protein in Ustilaginoidea virens, oligopeptide transporter in Colletotrichum gloeosporioides, PP2C in Fusarium oxysporum, have been implicated as pathogenicity genes. The availability of the diversified pathogenic mutants lay a new foundation for further study on the molecular pathogenic mechanisms of F. sacchari.
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| 科 Family | 属数 Number of genus | 种数 Number of species | 占总种数比例 Percentage of total species (%) | 属 Genus | 种数 Number of species | 占总种数比例 Percentage of total species (%) |
|---|---|---|---|---|---|---|
| 鹅膏菌科Amanitaceae | 2 | 11 | 5.26 | 鹅膏菌属 Amanita | 10 | 4.78 |
| 小菇科 Mycenaceae | 2 | 12 | 5.74 | 丝盖伞属 Inocybe | 5 | 2.39 |
| 多孔菌科 Polyporaceae | 8 | 14 | 6.70 | 蜡蘑属 Laccaria | 5 | 2.39 |
| 红菇科 Russulaceae | 3 | 23 | 11.00 | 小皮伞属 Marasmius | 6 | 2.87 |
| 小菇属 Mycena | 11 | 5.26 | ||||
| 光柄菇属 Pluteus | 5 | 2.39 | ||||
| 红菇属 Russula | 17 | 8.13 | ||||
| 栓菌属 Trametes | 5 | 2.39 |
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