Pathogenicity Differentiation of Fusarium spp. Causing Fusarium Basal Rot and Wilt Disease in Allium spp.

Here, 12 Fusarium strains, previously described as F. oxysporum f. sp. cepae (Foc), were examined via multi-locus sequencing of calmodulin (cmdA), RNA polymerase II second largest subunit (rpb2), and translation elongation factor 1-alpha (tef1), to verify the taxonomic position of Foc in the newly established epitype of F. oxysporum. The strains in this study were divided into two clades: F. nirenbergiae and Fusarium sp. To further determine the host specifications of the strains, inoculation tests were performed on onion bulbs and Welsh onion seedlings as potential hosts. Four strains (AC145, AP117, Ru-13, and TA) isolated from diseased onions commonly possessed the secreted in xylem (SIX)-3, 5, 7, 9, 10, 12, and 14 genes and were pathogenic and highly aggressive to onion bulbs, whereas all strains except for one strain (AF97) caused significant inhibition of Welsh onion growth. The inoculation test also revealed that the strains harboring the SIX9 gene were highly aggressive to both onion and Welsh onion and the gene was expressed during infection of both onions and Welsh onions, suggesting the important role of the SIX9 gene in pathogenicity. This study provides insights into the evolutionary pathogenicity differentiation of Fusarium strains causing Fusarium basal rot and wilt diseases in Allium species.

Effects of flusulfamide on spore germination of Plasmodiophora brassicae.

Flusulfamide inhibits germination of Plasmodiophora brassicae resting spores to suppress clubroot disease, but its mechanism of action on the germination of P. brassicae resting spores remains unclear. In this study, P. brassicae resting spores were treated with flusulfamide and visualized using transmission electron microscopy (TEM). The gene expression of P. brassicae resting spores was analyzed using RT-PCR, followed by immunoblotting analysis. TEM results revealed that flusulfamide suppressed the primary zoosporogenesis of P. brassicae resting spores during the early phase, and RT-PCR results revealed that flusulfamide affected the gene expression during the germination of the resting spores. Immunoblot and RT-qPCR analyses revealed that PbCyp3, an immunophilin (peptidyl-prolyl-isomerase) gene, was highly expressed, resulting in the unusual accumulation of PbCYP3 protein in P. brassicae resting spores immediately after treatment with flusulfamide. This suggests that flusulfamide may cause aberrant folding of proteins involved in primary zoosporogenesis, thereby inhibiting germination.

Pathogenicity chromosome of Fusarium oxysporum f. sp. cepae.

Fusarium oxysporum f. sp. cepae (Foc) is the causative agent of Fusarium basal rot disease in onions, which leads to catastrophic global crop production losses. Therefore, the interaction of Foc with its host has been actively investigated, and the pathogen-specific (PS) regions of the British strain Foc_FUS2 have been identified. However, it has not been experimentally determined whether the identified PS region plays a role in pathogenicity. To identify the pathogenicity chromosome in the Japanese strain Foc_TA, we initially screened effector candidates, defined as small proteins with a signal peptide that contain two or more cysteines, from genome sequence data. Twenty-one candidate effectors were identified, five of which were expressed during infection. Of the expressed effector candidates, four were located on the 4-Mb-sized chromosome in Foc_TA. To clarify the relationship between pathogenicity and the 4-Mb-sized chromosome in Foc_TA, nine putative 4-Mb-sized chromosome loss strains were generated by treatment with benomyl (a mitotic inhibitor drug). A pathogenicity test with putative 4-Mb-sized chromosome loss strains showed that these strains were impaired in their pathogenicity toward onions. Genome analysis of three putative 4-Mb-sized chromosome loss strains revealed that two strains lost a 4-Mb-sized chromosome in common, and another strain maintained a 0.9-Mb region of the 4-Mb-sized chromosome. Our findings show that the 4-Mb-sized chromosome is the pathogenicity chromosome in Foc_TA, and the 3.1-Mb region within the 4-Mb-sized chromosome is required for full pathogenicity toward onion.

The SIX5 Protein in Fusarium oxysporum f. sp. cepae Acts as an Avirulence Effector toward Shallot (Allium cepa L. Aggregatum Group).

Fusarium oxysporum f. sp. cepae (Foc) causes basal rot disease in Allium species, including onions (Allium cepa L.) and shallots (A. cepa L. Aggregatum group). Among Allium species, shallots can be crossbred with onions and are relatively more resistant to Foc than onions. Thus, shallots are considered a potential disease-resistant resource for onions. However, the mechanisms underlying the molecular interactions between shallots and Foc remain unclear. This study demonstrated that SIX5, an effector derived from Foc (FocSIX5), acts as an avirulence effector in shallots. We achieved this by generating a FocSIX5 gene knockout mutant in Foc, for which experiments which revealed that it caused more severe wilt symptoms in Foc-resistant shallots than the wild-type Foc and FocSIX5 gene complementation mutants. Moreover, we demonstrated that a single amino acid substitution (R67K) in FocSIX5 was insufficient to overcome shallot resistance to Foc.

Molecular Analysis of MgO Nanoparticle-Induced Immunity against Fusarium Wilt in Tomato.

Fusarium wilt, caused by Fusarium oxysporum f. sp. lycopersici (FOL), is a devastating soilborne disease in tomatoes. Magnesium oxide nanoparticles (MgO NPs) induce strong immunity against Fusarium wilt in tomatoes. However, the mechanisms underlying this immunity remain poorly understood. Comparative transcriptome analysis and microscopy of tomato roots were performed to determine the mechanism of MgO NP-induced immunity against FOL. Eight transcriptomes were prepared from tomato roots treated under eight different conditions. Differentially expressed genes were compared among the transcriptomes. The Kyoto Encyclopedia of Genes and Genomes enrichment analysis revealed that in tomato roots pretreated with MgO NPs, Rcr3 encoding apoplastic protease and RbohD encoding NADPH oxidase were upregulated when challenge-inoculated with FOL. The gene encoding glycine-rich protein 4 (SlGRP4) was chosen for further analysis. SlGRP4 was rapidly transcribed in roots pretreated with MgO NPs and inoculated with FOL. Immunomicroscopy analysis showed that SlGRP4 accumulated in the cell walls of epidermal and vascular vessel cells of roots pretreated with MgO NPs, but upon FOL inoculation, SlGRP4 further accumulated in the cell walls of cortical tissues within 48 h. The results provide new insights into the probable mechanisms of MgO NP-induced tomato immunity against Fusarium wilt.