The Testing of TiO 2 Photocatalityc Reaction to Inhibit of Botryotinia fuckeliana

We studied the antifungal activity of the titanium dioxide (TiO2) photocatalytic reaction, which shows antibacterial effects under UVA (315 400 nm) irradiation, against Botryotinia fuckeliana. Under the UVA irradiation of 1.0 mW/cm, growth of B. fuckeliana treated with TiO2 solution was inhibited 55% more than that treated with sterile distilled water (SDW). Its effectiveness was almost same as chlorothalonil, a commercial fungicide. Furthermore, even TiO2 solution at lower concentration showed antifungal activity by TiO2 photocatalytic reaction under UVA irradiation, since growth of B. fuckeliana treated with TiO2 solution at 8.5 × 10 mg/l was significantly decreased compared to that treated with SDW. These results suggested that the TiO2 photocatalytic reaction might be available as a novel fungicide against phytopathogens generated before harvesting such as B. fuckeliana.


Introduction
In recent years, consumers have shown a growing interest in food safety.To date, agricultural crops have been supplied stably and effectively by using chemical pesticides, but there may be a risk of mammalian toxicity and environmental pollution by the chemical pesticides.Such chemical products are a health hazard to farmers and consumers, and remain in the crops.The alternatives such as organic farming or pesticide-free production may be time-consuming and incur damage from the phytopathogens and pests.Titanium dioxide (TiO 2 ) has a photocatalytic reaction under UVA (315 -400 nm) irradiation and shows many excellent effects such as disinfection (Maness et al., 1999;Ibáñez et al., 2003;Kim et al., 2003;Maneerat and Hayata, 2006;Chawengkijwanich & Hayata, 2008) and decomposition (Maneerat et al., 2003), and can be used for a deodorant (Murakami et al., 2004) and water purification (Herreara Melian et al., 2000;Wist et al., 2002), but there are few reports on agricultural crops (Lu et al., 2006).When activated by UVA irradiation, the TiO 2 photocatalytic reaction generates hydroxyl radicals and superoxide ions at the TiO 2 surface.The hydroxyl radicals are used as a powerful oxidizing agent to convert organic pollutants absorbed on the TiO 2 surface into CO 2 and water vapor (Fujishima and Honda, 1972;Fujishima et al., 2000).In present study, we therefore compared the antifungal activity of the TiO 2 photocatalytic reaction on Botryotinia fuckeliana with common chemical pesticides.B. fuckeliana is one of the airborne phytopathogens with a necrotrophic lifestyle attacking over 200 crop hosts worldwide (Williamson et al., 2007).

Preparation of TiO 2 and Chlorothalonil Solutions
TiO 2 solution (8.5 × 10 2 mg/l Miracle titania M solution; a mixture of peroxotitanium acid solution and peroxo-modified anatase solution, Ohno Sekiyuten Co. Ltd., Hiroshima, Japan) was diluted with sterile distilled water (SDW) and used for the experiments.
Chlorothalonil (active ingredient: 40% tetrachloroisophthalonitrile, Sumitomo Chemical Garden Products Inc., Tokyo, Japan), a commercial fungicide, was diluted with SDW and used for the experiments as a positive control.

Measurements of Antifungal Activity
Botryotinia fuckeliana (NBRC9760) was purchased from the National Institute of Technology and Evaluation (Kisarazu, Japan).One ml of TiO 2 solution, SDW or chlorothalonil was spread on potato dextrose agar (PDA) plates, the surfaces of which were aseptically air-dried.A fraction of agar containing B. fuckeliana (0.4 cm in diameter) was plated at the center of the PDA plates (8.5 cm in diameter) and cultured at 25°C under UVA (315 -400 nm) irradiation from 20-W black light lamps for 7 days.The UVA irradiation intensity from the 20-W black light lamps was measured at 0.0 (no irradiation), 0.3, 0.5 and 1.0 mW/cm during the experiments by a UVR-400 radiometer (S-365 UV-sensor, wavelength sensitivity of 320 -410 nm, Iuchi, Osaka, Japan).After irradiation, the colony diameter of grew B. fuckeliana was measured with vernier calipers (Fig. 1).

Statistical Analysis
All experiments were performed in triplicate.The data presented are the means with standard errors.The experimental data were evaluated by analysis of variance (ANOVA), and the significant difference was analyzed using the Turkey-Kramer method at p‹0.05.

Results
Inhibitory effect of chlorothalonil on the colony growth of B. fuckeliana is shown in Figure 2. Antifungal activity increased with the concentration of chlorothalonil, and there was significantly antifungal activity at the concentration of chlorothalonil more than 1.0 × 10 2.6 mg/l.The level is commonly-used concentration.Therefore, antifungal activity of TiO 2 photocatalytic reaction against B. fuckeliana was compared with that of 1.0 × 10 2.6 mg/l chlorothalonil.
Inhibitory effect of UVA irradiation on the colony growth of B. fuckeliana by TiO 2 photocatalytic reaction is shown in Figure 3.With the SDW treatment, the growth of B. fuckeliana was inhibited 18% at 1.0 mW/cm 2 of UVA irradiation, although there were no significant differences in the growth at the range of UVA irradiation intensity less than 0.5 mW/cm 2 .In contrast, the growth of B. fuckeliana treated with TiO 2 was inhibited significantly under all levels of UVA irradiation.Growth of B. fuckeliana treated with TiO 2 solution under 0.3, 0.5, and 1.0 mW/cm 2 of UVA irradiation decreased approximately 34, 40, and 55%, respectively, compared to non-treated B. fuckeliana, which grew similarly under all levels of UVA irradiation intensity.The TiO 2 photocatalytic reaction showed a good ability to inhibit the growth of B. fuckeliana comparable to that of chlorothalonil at 1.0 × 10 2.6 mg/l, an adequate concentration, when used TiO 2 solution under 1.0 mW/cm 2 of UVA irradiation.
Inhibitory effect of TiO 2 at various concentrations on the colony growth of B. fuckeliana by TiO 2 photocatalytic reaction is shown in Figure 4.The antifungal activity of the TiO 2 photocatalytic reaction against B. fuckeliana increased concomitant with the concentration of TiO 2 solution.Growth of B. fuckeliana was inhibited approximately 55, 54, 48, 40, and 34% by photocatalytic reaction with TiO 2 solution diluted to 8.5 × 10 2 (no dilution), 8.5 × 10 1 , 8.5 × 10 0 , 8.5 × 10 -1 , and 8.5 × 10 -2 mg/l, respectively, compared to non-treated B. fuckeliana, which grew similarly under all levels of UVA irradiation intensity.These responses clarified that the TiO 2 photocatalytic reaction was highly efficient for inhibiting the growth of B. fuckeliana.

Discussion
The TiO 2 photocatalytic reaction has an antibacterial activity on many bacteria causing food poisoning (Kim et al., 2003;Kuhn et al., 2003).The antifungal activity of TiO 2 photocatalytic reaction against phytopathogens that might affect postharvest tomatoes and kiwifruit has also been tested (Hur et al., 2005;Maneerat and Hayata, 2006).Furthermore, inactivation of Fusarium oxysporum and Pythium aphanidermatum in hydroponic solution by TiO 2 photocatalytic reaction have been studied (Nakabayashi et al., 1999;Koohakan et al., 2003).In present study, the antifungal activity of TiO 2 photocatalytic reaction against B. fuckeliana was highly effective at UVA irradiation intensity more than 1.0 mW/cm 2 .The antifungal activity may be of great use in the agricultural field, since UVA irradiation intensity contained in the sunlight near the equator is approximately 2.0 mW/cm 2 (Joyce et al., 1996;Lonnen et al., 2005).The results of present study are suggested to have raised the possibility of using the TiO 2 photocatalytic reaction before harvesting at agricultural field.Moreover, phytopathogens may get tolerant on chemical bactericides, but appear to gain no resistance to the TiO 2 photocatalytic reaction, since the antibacterial activity of the TiO 2 photocatalytic reaction occurs with oxidation.TiO 2 is nontoxic and has been approved by the American Food and Drug Administration for use in human food, drugs, and cosmetics, and compounded in food contact materials such as cutting boards and other surfaces that come in contact with unprotected food.Furthermore, it is safe and nontoxic with excellent durability, and is used in toothpaste and cosmetics, and is approved as food additive (Taoda, 2009).

Conclusion
The TiO 2 photocatalytic reaction at UVA irradiation more than 1.0 mW/cm 2 exhibited a high antifungal activity comparable to that of chlorothalonil, a commercial fungicide.The effectiveness increased with the concentration of TiO 2 solution.It is suggested that TiO 2 is available as a novel fungicide against phytopathogens generated before harvesting such as B. fuckeliana.In future, to introduce TiO 2 photocatalytic reaction into actual agricultural field, it is necessary to do in vivo experiment.

Figure 3 .
Figure 3.Effect of UV irradiation on inhibiting the colony growth of B. fuckeliana by TiO 2 photocatalytic reaction Vertical bars at the top of the shaded bars indicate the standard error.Different letters indicate a significant difference (p‹0.05).

Figure 4 .
Figure 4. Effect of TiO 2 solution at various concentrations on inhibiting the colony growth of B. fuckeliana by TiO 2 photocatalytic reaction Vertical bars at the top of the shaded bars indicate the standard error.Different letters indicate a significant difference (p‹0.05).