RESUMO
Successive cultivation of fungi on artificial media has been reported to cause the sectorization, which leads to degeneration of developmental phenotype, and virulence. Fusarium oxysporum f. sp. niveum (Fon), the causal agent of watermelon Fusarium wilt, forms degenerated sectors after successive cultivation. In the present research, we demonstrated that subculture with aged mycelia increased the incidence of degenerations. To further investigate the differences between the Fon wild type (sporodochial type, ST) and variants (MT: mycelial type and PT: pionnotal type), developmental phenotypes and pathogenicity to watermelon were examined. Results in variants (PT2, PT3, PT11, and MT6) were different from ST with mycelia growth, conidia production and chlamydospore formation. Virulence of degenerated variants on susceptible watermelon Grand Baby (GB) cultivar was determined after inoculation with Fon variants and Fon ST. In root dipping methods, Fon variants showed no significant differences in disease progress compared with ST. Fon variants showed a significant decrease in disease progression compared with ST through infested soil inoculation. The contrasting results of two inoculation methods suggest that the degenerative changes due to repeated successive cultivation may lead to the loss of pathogen virulence-related factors of the early stage of Fon infection process. Therefore, cell wall-degrading enzymes (CWDEs; cellulase, pectinase, and xylanase) activities of different variants were analyzed. All Fon degenerated variants demonstrated significant decreases of CWDEs activities compared with ST. Additionally, transcript levels of 9 virulence-related genes (fmk1, fgb1, pacC, xlnR, pl1, rho1, gas1, wc1, and fow1) were assessed in normal state. The degenerated variants demonstrated a significantly low level of tested virulence-related gene transcripts except for fmk1, xlnR, and fow1. In summary, the degeneration of Fon is triggered with successive subculture through aged mycelia. The degeneration showed significant impacts on virulence to watermelon, which was correlated with the reduction of CWDEs activities and declining expression of a set of virulence-related genes.
RESUMO
Fusarium wilt, caused by Fusarium oxysporum (Fo), is one of the most important fungal diseases worldwide. Like other plant pathogens, Fo displays specialized forms in association with its hosts. For example, F. oxysporum f. sp. niveum (Fon) is the damaging pathogen causing Fusarium wilt disease on watermelon, whereas F. oxysporum f. sp. cubense is the pathogen that infects banana. A rapid and reliable pathogen identification or disease diagnosis is essential for the integrated disease management practices in many crops. In this study, two new primer sets, Fon-1/Fon-2 and FnSc-1/FnSc-2, were developed to differentiate Fon and Fo, respectively. The PCR method using the novel primer sets has high sensitivity to detect Fon when the DNA concentration was as low as 0.01 pg or when the conidia number was as few as 5. In comparison with the published primer set, the Fon-1/Fon-2 primer set, derived from the sequence of OP-M12 random primer-amplified fragment, produced a 174 bp DNA fragment, and was more specific to Fon in Taiwan. In addition, with optimized PCR parameters, the molecular method using the Fon-1/Fon-2 primer set could directly detect Fon even when watermelon samples were collected in its early stage of disease development.
