Effects of flusulfamide on spore germination of Plasmodiophora brassicae

Abstract

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.

Keywords: clubroot; flusulfamide; immunophilin; primary zoosporogenesis; resting spores.

Fig. 1. Transmission electron microscopy (TEM). (A) TEM images showing different stages of primary zoosporogenesis of P. brassicae resting spores. Numerals 0, 1, 2, and 3 correspond to the stages of primary zoosporogenesis. The following annotations are used: s, space between the cell wall and the cell membrane; v, vacuole; ve, vesicle containing an electron-dense granule; ms, microvillus-like vesicle. Black circles in the cytoplasm of resting spores indicate lipid bodies. (B) Percentage of the four stages (0, 1, 2, and 3) of primary zoosporogenesis of P. brassicae resting spores. The four stages of primary zoosporogenesis at 3- and 6-days post-incubation (DPI) were determined by quantifying TEM images. P. brassicae resting spores were incubated in distilled water (DW), distilled water containing flusulfamide (1 ppm) (DW+FS), cabbage hydroponic solution (CHS), or cabbage hydroponic solution containing flusulfamide (1 ppm) (CHS+FS). Numerals 0, 1, 2, and 3 represent the stages of primary zoosporogenesis of P. brassicae resting spores in each treatment group.

Fig. 2. Effect of flusulfamide on the expression of immunophilin genes. (A) RT-qPCR analyses of 20 immunophilins (11 cyclophilins (PbCYP), seven FK506 binding proteins (FKBP), and two parvulin-like proteins (PAR) genes in P. brassicae resting spores treated with flusulfamide for 24 hr. Values represent means±S. E. Statistical significance was determined using Student’s t-test. Asterisks indicate significant differences between P. brassicae resting spores treated with water (control) and flusulfamide (1 ppm); *p<0.05 and **p<0.01. (B) Time–course gene expression analyses of three cyclophilin genes: PbCYP3 (open circles), PbCYP8 (filled squares), and PbCYP10 (open squares). Control (filled circles) corresponds to the reference gene (PbACT1) used for RT-qPCR normalization. Values represent means±S. E. Statistical significance was determined using Student’s t-test. Asterisks indicate significant differences between P. brassicae resting spores treated with water (control) and flusulfamide (1 ppm); *p<0.05 and **p<0.01. (C) Silver staining of SDS-PAGE (left) and immunoblot analysis (right) of proteins extracted from P. brassicae resting spores treated with flusulfamide for 24 hr: anti-PbCYP3 IgG was used as the primary antibody. C, flusulfamide-untreated; F, flusulfamide-treated.