JIA-2018-09

2025 Tamaki Masahiko et al. Journal of Integrative Agriculture 2018, 17(9): 2024–2030 rice production has increased. However, eutrophication is a direct consequence of this trend, with high concentrations of nitrogen and phosphorus in agricultural area, subsequently affecting aquatic organisms. As consumers have become more aware of the negative effects of pesticide use, rice cultivated with reduced pesticide usage is traded at higher prices. Recently, tiny bubbles less than 50 µm in diameter, so-called “microbubbles” (MB), have been studied and used in many fields. The characteristic of MB includes slow ascent rate, self-pressurization, high gas solubility, high surface potential and the production of free radicals by collapsing (Takahashi 2005). In agricultural fields, the inactivation of Fusarium oxysporum f. sp. melonis spores and Pectobacterium carotovorum subsp. carotovorum as well as removal of residual pesticide in fruits and vegetables by ozone MB have been investigated; further studies have also examined pasteurization with pressurized carbon dioxide microbubbles (CO 2 MB) (Kobayashi et al . 2009, 2011; Ikeura et al . 2013). Moreover, the free radicals generated from CO 2 MB has been associated with the inactivation of Pseudomonas putida (Mulakhudair et al . 2017). In addition, MB intensify the cleaning effect of surfactants (Takagi 2006), although the underlying mechanism is still unclear. In this context, we investigate the usage of Blasin ® Flowable (BF), a disinfectant for inhibiting rice blast. In particular, the inhibition effect of BF solution containing CO 2 MB on the germination and appressorium formation of P . oryzae Cavara conidia was examined; CO 2 was used as it was a harmless and inexpensive gas. In addition, the effect of BF solution containing CO 2 MB on P . oryzae Cavara conidia was investigated with electron microscopy. 2. Materials and methods 2.1. Preparation of conidial suspension of P. oryzae Cavara The conidial suspension of P . oryzae Cavara was prepared as follows: the P . oryzae Cavara strain Hoku-1 (NBRC30736) was obtained from the Laboratory of Plant Pathology at Meiji University (Kawasaki, Japan). A fraction of agar containing P . oryzae Cavara was plated on oatmeal agar (OMA, 50 g of oatmeal, 20 g of sucrose and 20 g of agar in 1 L of distilled water), an agar for forming P . oryzae conidia, and incubated at 30°C for 20 days in the dark. After incubation, the colonies were exposed to near-ultraviolet black light (300–450 nm, 10 W (50 Hz), Tarutanidenki Co., Ltd., Higashi-Osaka, Japan) from a height of 30 cm for 2–3 days (Ohmori and Nakajima 1970). After irradiation, 14 mL of distilled water was poured on the plate, and the surface of the plate was lightly scraped with a spatula. The obtained solution was filtered through double gauze to remove any hyphae and conidiophores from the solution. The number of conidia in the solution was measured with a Thoma Counting Chamber (Hemacytometers no. A4200, Fujirika Co., Ltd., Osaka, Japan) and prepared to a concentration of 6.0×10 5 spores mL –1 with distilled water. The P . oryzae Cavara conidia suspended in distilled water were used for all experiments in this study. Based on previous studies, the appressorium formation ratios in distilled water, glucose or peptone solution are similar (Xiao et al . 1994). 2.2. Preparation of the Blasin ® Flowable solution The BF solution (Hokko Chemical Industry Co., Ltd., Tokyo, Japan) diluted to 1000-, 5000-, 10000- and 50000-fold with tap water, was used for the experiment. It contains 15% Fthalide and 15% Ferimzone as active ingredients. Fthalide inhibits the reductase activity of the main metabolic pathway in the pathogen, blocking the biosynthesis of melanin and preventing the pathogen from making progress after appressorium formation (Chida 1989). Ferimzone prevents the pathogen from transporting acetic acid and pyrvic acid into the hypha and influences membrane function involved in the permeability of acid electrolytes such as hydrogen ions. Consequently, hyphal growth is inhibited due to leakage of acid electrolyte from the hypha (Okuno et al . 1989). 2.3. Treatment of P. oryzae Cavara conidium by CO 2 MB-treated BF solution The CO 2 MB was generated with decompression-type (FS 101-L1OZ; Fuki Co., Ltd., Kumagaya, Japan) and the gas- water circulating-type (20NPD04S; Shigenkaihatsu Co., Ltd., Yokohama, Japan) MB generators in a cylindrical container (φ31.0 cm×61.0 cm, 42 L) containing 20 L of BF solution (20°C) for 5 min. It was quantitatively confirmed that the amounts of Fthalide and Ferimzone in the BF solution were not decreased by MB treatment (data not shown). For comparison, CO 2 millibubbles (CO 2 MMB) treatment was performed by feeding CO 2 using an air stone and pump. The pH of the CO 2 MB-treated BF solution was measured with a pHmeter (D-51, Horiba Ltd., Kyoto, Japan). A total of 1 mL of CO 2 MB-treated BF solution was then added to a 1.5-mL tube containing 0.1 mL of P . oryzae Cavara conidial suspension and mixed with a vortex mixer. Subsequently, 10 mL of the mixture was dispensed on a polycarbonate board and left to stand at 20°C for 24 h in the dark at high humidity. 2.4. Measurement of germination and appressorium formation ratios of P. oryzae Cavara conidia Six viewing fields per sample were observed with a