JIA-2018-09

2026 Tamaki Masahiko et al. Journal of Integrative Agriculture 2018, 17(9): 2024–2030 fluorescence microscope (×200, BZ-9000; Keyence Co., Osaka, Japan). A polycarbonate board, which showed the best germination and appressorium formation ratios of P . oryzae Cavara conidia in artificial materials (data not shown), was selected as inoculating material. The numbers of total conidia, germinated conidia that visually confirmed the extension of germ tube more than 3 mm, and appressorium-forming conidia were determined in each viewing field. The percentages of germination and appressorium formation ratios were calculated, and means and standard errors of six viewing fields are presented as result. 2.5. Observation of P. oryzae Cavara conidia by electron microscopy Observation of P . oryzae Cavara conidia by electron microscopy was performed (Kobayashi et al . 2014). Conidia were exposed to 5000-fold diluted BF solution treated with CO 2 MMB and CO 2 MBs for 5 min. For comparison, the conidia were also exposed to 5000-fold diluted BF solution. Subsequently, 1 mL of the treated P . oryzae Cavara conidial solution was dispensed into a 1.5-mL tube and the conidia were collected by double centrifugation (6 000 r min –1 , 5 min) and re-suspended in 1/15 mol L –1 phosphate buffer solution (pH 7.0). After that, the solution was mixed with 25% glutalaldehyde (Kanto Chemical Co., Inc., Tokyo, Japan) (final concentration of glutalaldehyde was 2.5%), pre-fixed at room temperature for 1 h and post-fixed by 2%OsO 4 solution at room temperature for 1 h with subsequent dehydration in 50, 70, 80, 90, 95 and 99.5% ethanol solution (the ethanol solution concentration was set up with phosphate buffer solution). Observation by scanning electron microscopy (SEM) was performed as follows: The dehydrated samples were immersed in a mixture of t -butyl alcohol and dehydrated ethanol (1:1); after 10 min, it was placed in 100% t -butyl alcohol and frozen overnight at –20°C. The samples were then freeze-dried with a freeze drier (ES-2030, Hitachi High Technologies Co., Tokyo, Japan) and OsO 4 -coated with an OsO 4 coater (HPC-1SW, Vacuum Device Inc., Mito, Japan) for 10 s under vacuum (coating thickness was adjusted to 3 nm). The samples were then observed with SEM (JSM- 6700F, JEOL Ltd., Akishima, Japan), operated at 3 kV. Observation by transmission electron microscopy (TEM) was performed as follows: The dehydrated samples were immersed in a mixture of Quetol-651 (Cosmo Bio Co., Ltd., Tokyo, Japan) and ethylene glycol diglycidyl ether (1:1) and kept for 1 h. The samples were then serially replaced in the mixtures (a ratio of 2:1 and 3:1) and 100% Quetol-651 and embedded at 60°C for 48 h. Ultra-thin sections (90 nm thickness) were obtained from the embedded samples, using an ultramicrotome (ULTRA CUT UCT, Leica Microsystems, Wetzlar, Germany). The sections were doubly electron- strained with 4% uranyl acetate solution for 12 min and lead nitrate solution for 5 min and then observed with TEM (JEM-2010, JEOL Ltd.) operated at 140 kV. 2.6. Statistical analysis The statistical significance of differences was evaluated by the Tukey-Kramer test ( P <0.01). 3. Results The effects of different concentrations of BF solutions containing CO 2 MMB and both CO 2 MBs on the inhibition of germination and appressorium formation of P . oryzae Cavara conidia are shown in Fig. 1. The germination ratio of the conidia treated with BF solution containing the decompression-type CO 2 MB was significantly lower than that with CO 2 MMB, the gas-water circulating-type CO 2 MB and nontreatment at 10000-fold dilution, and was the same as the gas-water circulating-type CO 2 MB and CO 2 MMB at 5 000-fold dilution but was significantly less than that of nontreatment. Germination of P . oryzae Cavara conidia could be completely inhibited by 1 000-fold diluted FB solution containing both CO 2 MBs, but not CO 2 MMB. The appressorium formation of P . oryzae Cavara conidia of 10 000-fold diluted FB solution containing both CO 2 MBs was significantly lower than that of CO 2 MMB and nontreatment. The complete inhibition could be achieved by 5 000-fold diluted FB solution containing both CO 2 MBs and by 1 000-fold diluted FB solution containing CO 2 MMB and both CO 2 MBs. The SEM images of P . oryzae Cavara conidia treated with BF solution containing CO 2 MMB and both CO 2 MBs are shown in Fig. 2. A cancellous pattern was observed for the conidia in nontreatment (Fig. 2-A). In contrast, the conidia treated with BF solution containing both CO 2 MBs (Fig. 2-D and E) showed deeper wrinkles; the damage on the surface of the conidia treated with BF solution containing the decompression-type CO 2 MB was higher than that containing the gas-water circulating-type CO 2 MB. Wrinkles were also confirmed on the surface of the conidia treated with BF solution containing CO 2 MMB, although the degree was lower than that for conidia treated with BF solution containing both CO 2 MBs. The width of the conidium treated with BF solution containing the decompression-type CO 2 MB was smaller than that of conidia treated with BF solution containing CO 2 MMB, although the length was unchanged. The TEM images of P . oryzae Cavara conidia treated with BF solution containing CO 2 MMB and both CO 2 MBs are shown in Fig. 3. The TEM images of the conidia in