Tamaki Masahiko, Kobayashi Fumiyuki, Suehiro Keisuke, Ohsato Shuichi, Sato Michio

1 Schoolof Agriculture, Meiji University, Kawasaki 214-8571, Japan

2 Faculty of Applied Life Science, Nippon Veterinary and Life Science University, Musashino 180-8602, Japan

Abstract We investigated the possibility to reduce the usage of Blasin®Flowable (BF), a disinfectant inhibiting the germination and appressorium formation of Pyricularia oryzae Cavara conidia, by using carbon dioxide microbubbles (CO2MB). Germination was significantly inhibited by 10 000-fold diluted BF solution containing CO2MB generated by the decompression-type generator compared to CO2 millibubbles (CO2MMB) and CO2MB generated by the gas-water circulating-type generator.Appressorium formation in the 10 000-fold diluted BF solution containing both CO2MBs was less than that in CO2MMB.Scanning electron microscopy showed wrinkles and dents on the surface of conidia treated with 5 000-fold diluted BF solution containing both CO2MBs. Via transmission electron microscopy, we observed the expansion of the vacuole and the intracellular space and bloated or absent lipid granules in the conidia treated with BF solution containing both CO2MBs.Our results show that inhibition of the conidium germination and appressorium formation of P. oryzae Cavara by 10 000-fold diluted BF solution could be achieved by using the decompression-type CO2MB.

Keywords: appressorium formation, Blasin®Flowable, germination, Pyricularia oryzae, carbon dioxide microbubble

1. Introduction

Rice blast is the most important disease in Japanese rice cultivation. With improved rice cultivation techniques,yields per 10 acres exceed 500 kg in Japan, with a steadily increasing surplus (MAFF 2015). However, yield deficits may occur in summer of low temperature and following the outbreak of rice blast. Pyricularia oryzae Cavara is a plant-pathogenic fungus that causes the development of rice blast; it grows steadily at periods with low temperature, poor sunshine and relatively heavy rain. The conidia germinate in the presence of water on the surface of paddy and forms an appressorium, with the elongation of the germ tube and a melanin layer just below the cell wall containing the septum (Bourett and Howard 1990). The hyphae expand in the plant from the stoma or by penetrating the cell wall,and several conidia are produced at the top. Hereby, the conidia on the lesion move to the rice stalk, affecting the stalk and the branch and subsequently causing necrosis in most rice tissues. Consequently, the rice stalk lacks nutrients and turns white, resulting in significant yield loss. To mitigate this problem, the use of chemical pesticides in Japanese rice production has increased. However, eutrophication is a direct consequence of this trend, with high concentrations of nitrogen and phosphorus in agriculturalarea, 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 manyfields. The characteristic of MB includes slow ascent rate, self-pressurization, high gas solubility,high surface potentialand the production offree radicals by collapsing (Takahashi 2005). In agriculturalfields, the inactivation of fusarium oxysporum f. sp. melonis spores and Pectobacterium carotovorum subsp. carotovorum as wellas removalof residual pesticide in fruits and vegetables by ozone MB have been investigated; further studies have also examined pasteurization with pressurized carbon dioxide microbubbles (CO2MB) (Kobayashi et al. 2009, 2011;Ikeura et al. 2013). Moreover, the free radicals generated from CO2MB 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 stillunclear.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 CO2MB on the germination and appressorium formation of P. oryzae Cavara conidia was examined; CO2was used as it was a harmless and inexpensive gas. In addition, the effect of BF solution containing CO2MB 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 oatmealagar (OMA, 50 g of oatmeal, 20 g of sucrose and 20 g of agar in 1 lof 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 mlof distilled water was poured on the plate, and the surface of the plate was lightly scraped with a spatula. The obtained solution wasfiltered 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 6.0×105spores mL-1with 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 1 000-, 5 000-, 10 000- and 50 000-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 in fluences 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 CO2MB-treated BF solution

The CO2MB was generated with decompression-type (FS 101-L1OZ; Fuki Co., Ltd., Kumagaya, Japan) and the gaswater circulating-type (20NPD04S; Shigenkaihatsu Co., Ltd.,Yokohama, Japan) MB generators in a cylindrical container(φ31.0 cm×61.0 cm, 42 L) containing 20 lof BF solution(20°C) for 5 min. It was quantitatively confirmed that the amounts offthalide and Ferimzone in the BF solution were not decreased by MB treatment (data not shown). For comparison, CO2millibubbles (CO2MMB) treatment was performed by feeding CO2using an air stone and pump. The pH of the CO2MB-treated BF solution was measured with a pH meter (D-51, Horiba Ltd., Kyoto, Japan). A totalof 1 mlof CO2MB-treated BF solution was then added to a 1.5-mL tube containing 0.1 mlof P. oryzae Cavara conidial suspension and mixed with a vortex mixer. Subsequently, 10 mlof 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 viewingfields per sample were observed with a 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 viewingfield. The percentages of germination and appressorium formation ratios were calculated, and means and standard errors of six viewingfields 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 5 000-fold diluted BF solution treated with CO2MMB and CO2MBs for 5 min. For comparison, the conidia were also exposed to 5 000-fold diluted BF solution.Subsequently, 1 mlof 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-1phosphate 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% OsO4solution 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-butylalcoholand dehydrated ethanol (1:1); after 10 min, it was placed in 100% t-butylalcoholand frozen overnight at -20°C. The samples were then freeze-dried with a freeze drier (ES-2030, Hitachi High Technologies Co., Tokyo, Japan) and OsO4-coated with an OsO4coater (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 electronstrained with 4% uranylacetate 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. Statisticalanalysis

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 CO2MMB and both CO2MBs 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 CO2MB was significantly lower than that with CO2MMB, the gas-water circulating-type CO2MB and nontreatment at 10 000-fold dilution, and was the same as the gas-water circulating-type CO2MB and CO2MMB 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 CO2MBs, but not CO2MMB. The appressorium formation of P. oryzae Cavara conidia of 10 000-fold diluted FB solution containing both CO2MBs was significantly lower than that of CO2MMB and nontreatment.The complete inhibition could be achieved by 5 000-fold diluted FB solution containing both CO2MBs and by 1 000-fold diluted FB solution containing CO2MMB and both CO2MBs.

The SEM images of P. oryzae Cavara conidia treated with BF solution containing CO2MMB and both CO2MBs 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 CO2MBs(Fig. 2-D and E) showed deeper wrinkles; the damage on the surface of the conidia treated with BF solution containing the decompression-type CO2MB was higher than that containing the gas-water circulating-type CO2MB. Wrinkles were also confirmed on the surface of the conidia treated with BF solution containing CO2MMB, although the degree was lower than that for conidia treated with BF solution containing both CO2MBs. The width of the conidium treated with BF solution containing the decompression-type CO2MB was smaller than that of conidia treated with BF solution containing CO2MMB, although the length was unchanged.

Fig. 1 Inhibition of germination and appressorium formation of Pyricularia oryzae Cavara conidia by Blasin®Flowable (BF) solution containing carbon dioxide millibubbles (CO2MMB) and both CO2 microbubbles (CO2MBs). Treatment with BF solution was performed for 5 min. A, germination ratio. B, appressorium formation ratio. Different uppercase and lowercase letters indicate significant differences among the BF concentration of the same treatments and different treatments at the same concentration of BF by Tukey-Kramer method, respectively (P＜0.01). Data are means±SE (n=6).

The TEM images of P. oryzae Cavara conidia treated with BF solution containing CO2MMB and both CO2MBs are shown in Fig. 3. The TEM images of the conidia in nontreatment (Fig. 3-A), the treatment with FB solution only(Fig. 3-B) and containing CO2MMB (Fig. 3-C) show that some conidia had lipid granules lining up along the lines inside the membrane and the septum; however, the conidia treated with FB solution containing both CO2MBs (Fig. 3-D and E) showed bloated or no lipid granules and an expansion of the vacuole and the intracellular space. Furthermore,intracellular space site of the conidium treated with BF solution containing the decompression-type CO2MB were damaged worse than the gas-water circulating-type CO2MB.

4. Discussion

According to Takahashi (2005), MB has the larger specific surface area and the slower ascend rate than MMB, resulting in higher gas dissolution efficiency. In the present study,the dissolved CO2concentration in both CO2MBs was higher than that in CO2MMB because the pH of the BF solution treated with both CO2MBs was lower than that with CO2MMB. A previous study has shown that the inactivation of Bacillus cereue spores by heat treatment can be promoted by dissolved CO2and decreased pH, which can be achieved with the addition of CO2(Kim et al. 2012). Takagi (2006)also stated that MB led to a drastic decrease in the ascend rate and to coalescence inhibition and sterilization over a prolonged period in the presence of surfactants. In addition,the MB size decreases in the presence of surfactants(Fujioka et al. 2012), while physicalabsorption and matter transport capacity increase in the characteristic gas-liquid interface of MB (Li et al. 2009). Therefore, we consider that the contact between P. oryzae Cavara conidia and CO2MB increased due to the stabilization and the decrease of ascend rates in the presence of BF, resulting in decrease of germination ratios.

In addition, the shrinkage of MB caused by gas dissolution increases the inner pressure and the surface area, and a logarithmic increase in the shrinkage rate has been observed. Hydroxyl radicals are generated due to the decomposition of water molecules, associated with the collapse of MB during shrinkage, and directly attack the DNA,enzymes and other components of microorganisms, leading to cell death (Takahashi 2005). Takahashi et al. (2007) also showed the decomposition of phenol due to hydroxyl radicals produced by collapsing AirMB. Mulakhudair et al. (2017)suggested that the inactivation of P. putida was caused by the free radicals generated from CO2MB. Similarly,Kukizaki and Baba (2008) indicated that the size of the MB generated in an anionic surfactant solution was smaller than that of MB generated in a non-ionic surfactant solution, and a strong electrostatic repulsion between MB was induced.Because anionic and non-ionic surfactants were contained in the BF solution used in the present experiment, it might be a possibility to increase hydroxyl radical production with increasing anionic charge during MB treatment. Based on our results, not only dissolved CO2concentration and the low pH but also the generation of hydroxyl radicals had an effect on the inhibition of appressorium formation of P. oryzae Cavara conidia.

The destruction or modification of the cell wall by the decompression-type CO2MB is more severe than that caused by CO2MMB and the gas-water circulating-type CO2MB (Fig. 2-C, D and E); this can be explained by the smaller size of the decompression-type CO2MB and the greater generation of hydroxyl radicals compared to the gas-water circulating-type CO2MB (Ikeura et al. 2017). In a similar study, MB promoted the breaking of algal cells by microwave treatment (Krehbiel et al. 2014). Specifically,it is presumed that MB directly act on the cell walland penetrate the conidia because of the high permeability. The reduction of the width, the void space and the absent lipid granules in the conidia were caused by treatment with BF solution containing both CO2MBs (Fig. 3-D and E). This may be due to the expansion of the vacuole by increasing the permeability of BF into the conidia by CO2MB, because Fthalide and Ferimzone, the active ingredients in BF, affect the metabolism of P. oryzae Cavara conidium (Chida 1989;Okuno et al. 1989). However, the length of the conidia was unchanged because they are composed of three cells and two septa.

5. Conclusion

In the present study, the concentration of BF solution required to inhibit germination and appressorium formation of P. oryzae Cavara conidia could be reduced by using CO2MB. Such a reduction in pesticide use is highly beneficial for the sustainable management of agriculturalfields and reduces the amount of residual pesticides in the environment. Based on electron microscopic observation,it could be confirmed that physicalalterations of the conidia,such as wrinkles, dents and decrease of the width, were caused by the use of BF solution containing CO2MB. This infers that the significant inhibition of germination and appressorium formation by BF solution containing CO2MB was due to the synergistic effect of CO2MB and the high concentration of BF. However, further investigations are needed to understand the underlying mechanisms.