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.

SUMMARY: Fusarium wilt of watermelon, caused by Fusarium oxysporum f. sp. niveum, is one of the limiting factors for watermelon production in Taiwan. In recent research, the phenylalanine ammonia lyase (PAL) gene expressed in the shoot base of the Fusarium wilt resistant line JSB was related to Fusarium wilt resistance. Phenylalanine ammonia lyase is the key regulatory enzyme in the phenylpropanoid metabolic pathway. The downstream products of phenolic compounds are considered to be involved in the complicated plant defence mechanisms. They could act as signal molecules, antimicrobial substances and/or structural barriers. To study the resistant mechanisms of Fusarium wilt, the resistant JSB line was examined for comparison of F. oxysporum-watermelon interactions with the susceptible Grand Baby (GB) cultivar. Unlike infected GB, which was seriously colonized by F. oxysporum in the whole plant, the pathogen was limited below the shoot base of inoculated JSB, suggesting that the shoot base of JSB may contribute to Fusarium resistance. The data indicated that a significant increase in PAL activity was found in shoot bases of the resistant JSB line at 3, 9, 12 and 15 days after inoculation (DAI). Shoot bases of resistant watermelons accumulated higher amounts of soluble and cell wall-bound phenolics at 3–9 DAI; the susceptible GB cultivar, however, only increased the cell wall-bound phenolics in shoot bases at 3 DAI. High lignin deposition in the cell walls of vascular bundles was observed in the shoot bases of JSB but not of GB seedlings at 6 and 9 DAI. In the roots and shoot bases of JSB seedlings at 6 DAI, peroxidase enzyme activity increased significantly. In summary, the results suggest that accumulation of cell wall-bound phenolics and increase of peroxidase activity in shoot bases of JSB seedlings during F. oxysporum inoculation, together with the rapid deposition of lignin in the cell walls of vascular bundles, may have provided structural barriers in resistant JSB line to defend against F. oxysporum invasion.

Plant-parasitic nematodes infect a diversity of crops, resulting in severe economic losses in agriculture. Microbial volatile organic compounds (VOCs) are potential agents to control plant-parasitic nematodes and other pests. In this study, VOCs emitted by a dozen bacterial strains were analyzed using solid-phase microextraction followed by gas chromatography–mass spectrometry. Fumigant toxicity of selected VOCs, including dimethyl disulfide (DMDS), 2-butanone, 2-pentanone, 2-nonanone, 2-undecanone, anisole, 2,5-dimethylfuran, glyoxylic acid, and S-methyl thioacetate (MTA) was then tested against Caenorhabditis elegans. DMDS and MTA exhibited much stronger fumigant toxicity than the others. Probit analysis suggested that the values of LC₅₀ were 8.57 and 1.43 μg/cm³ air for DMDS and MTA, respectively. MTA also showed stronger fumigant toxicity than DMDS against the root-knot nematode Meloidogyne incognita, suggesting the application potential of MTA.