Vegetative Compatibility Analysis For Fusarium Species Caused Sugarcane Pokkah Boeng

Sugarcane pokkah boeng caused by the filamentous fungus Fusarium species complex (F. verticillioides, F. proliferatum and F. oxysporum), is becoming a devastating disease in China sugarcane industry. Pokkah boeng disease has been recorded in almost all countries where sugarcane is grown commercially. To descipher the Fusarium species complex causing sugarcane pokkah boeng,115 strains were isolated from sugarcane production area throughout China. The genetic diversity of these strains complex was analyzed by PCR amplification of species-specific genes. The T-DNA inserational mutant library were generated by Agrobacterium tumerfaciens mediated transformation (ATMT) and their pathogenicity were evaluated by clony characterization and in vitro test. Vegetative compatibility analysis were carried out on these strains and mutants to describe the population structure, the differentiation and the epidemic regularity of the Fusarium caused sugarcane pokkah boeng. The clustering results from 5 pairs of species-specific primers showed that 115isolates were classified into 7 genetic groups. Group-Ⅰindued the isolates from Guangxi and Rongwei, Fujian. All isolates from Fuzhou Fujian were classified in Group-Ⅲ; nine isolates from F. proliferaturn in group-Ⅳ. Almost all isolates from Yunnan were clustered into three groups (Group-Ⅴ, Ⅶ and V-Ⅲ). The results indicated the abundant genetic diversity of Fusarium species caused sugarcane pokkah boeng. Each two isolates from the 7 genetic groups (in total of 14 isolates) were used for the vegetative compatibility analysis. After growing on the potassium chlorate medium, all isolates were able to form sectors, i.e., colonies fast growing at the tips of thickening hyphae, indicating that nit-mutants (nitrate non-utilizing mutants) had been formed. The different number of Nit mutants was produced in different Fusarium isolates and species. More than 80% of Nit 1 mutations were produced from the tesed isolates. However, only few NitM mutation were produced.These nit-mutants were then moved to the minimal media (MM) to be used for VCG analysis. From VCGs of 14 isolates identified, only YN49 and YN56 belonged to same VCG10, the other isolates had different VCGs. Therefore, the isolates from the Fusarium species complex caused sugarcane pokkah boeng showed abundant VCGs. These VCGs were not associated with geographic origin, host plants, species and their genetic diversity of our isolates.Total 667 T-DNA insertional mutants were generated from CNO-1 by Agrobacterium tumerfaciens mediated transformation (ATMT) and their pathogenicity were tested in vitro. The results indicated that most of mutants had pathogenicity ranged from Class 2 to Class 3. Three mutants (636B、41-1 和 41-2) with Class I decreased their pathogenicity, and 5 mutants (280、43-1、14、295 and 283) with Class V increased their pathogenicity. Eight mutants had more than 80% and only 3 had less than 30% in DSI (Disease Severity Index). After growing on the potassium chlorate medium, more than 60% of Nit 1 mutations were produced from the T-DNA insertional mutants, while 20% of the NitM mutation were produced from all T-DNA mutants. The NitM had strong compability to Nitl and Nit 3 from same T-DNA insertional mutants. The results from VCGs analysis showed that only mutant 280 were not compabile to other T-DNA insertional mutants, including 9 mutants with increased pathogenicity and three less pathogenicity. While VCG has no closed related to pathogenicity, it will provides a new trait to further characterize fungal strains. More importantly, these results suggest the existence of genetic limits that could govern paths of gene flow associated the orgin of the Fusarium pathogen caused sugarcane pokkah boeng.