Establishment of Rice Bakanae Disease Management Using Slightly Acidic Hypochlorous Acid Water

미산성 차아염소산수를 이용한 벼키다리 병 방제

Abstract

Rice bakanae is a typical seed-borne and seed-transmitted disease caused by infection by Fusarium fujikuroi. Seed disinfection using chemical fungicides (such as benomyl and prochloraz) is most effective in controlling the disease, but the emergence of fungicide-resistant strains has recently been increasing. Slightly acidic hypochlorous acid water (SAHW) is a safe and environmentally friendly disinfectant that has a potent and broad spectrum of antimicrobial activity against viruses, bacteria, and fungi. In this study, we aimed to investigate the effectiveness of SAHW against F. fujikuroi strains, including chemical fungicide-resistant strains, as an alternative to conventional chemical fungicides in the management of bakanae disease. SAHW showed strong but similar levels of antifungal activity among the F. fujikuroi strains with a minimum inhibitory concentration (MIC₉₀) of 5±2.5 ppm of free available chlorine (FAC). In addition, F. fujikuroi cells lost viability completely within 5 min of SAHW treatment due to the lethal damage to cell integrity. When the rice seeds infected by F. fujikuroi were treated with SAHW containing 20±10 ppm of FAC for 12 hr, the efficiencies of seed disinfection and disease control were 95-98% and 90.1-92.6%, respectively. Altogether, our data suggest that SAHW is an effective compound for controlling rice bakanae disease.

벼키다리병은 Fusarium fujikuroi의 감염에 의해 발생하는 대표적인 종자전염성 병이다. 이 병의 방제는 화학약제를 이용한 종자소독이 가장 효율적이지만 최근 약제에 저항성을 갖는 균주의 출현이 증가하고 있다. 미산성 차아염소산수는 환경이나 사람의 건강에는 유해성이 낮지만 바이러스, 박테리아, 곰팡이에 강력하고 광범위한 항균활성을 갖는 살균소독제로 알려져 있다. 본 연구는 화학 약제 저항성 또는 민감성 균주를 포함하는 16개의 F. fujikuroi 균주를 대상으로 미산성 차아염소산수의 항균활성을 조사하고, 이를 이용한 친환경적 병 방제 기술 개발을 목적으로 하였다. 미산성 차아염소산수는 화학약제 저항성 균주를 포함한 조사한 F. fujikuroi 균주 모두에 대해 유리염소 5±2.5 ppm 농도에서 비특이적이고 강한 항균 활성을 나타냈고, 처리 후 5분 이내에 F. fujikuroi 세포의 치명적 손상과 함께 완전한 살균 효과를 나타났다. F. fujikuroi 균주로 감염된 벼 종자를 20±10 ppm의 유리염소를 포함하는 미산성 차아염소산수로 12시간 침지 소독하였을 경우, 종자 소독 효과와 병 방제율은 각각 95~98%와 90.1~92.6%였다.

Introduction

Rice bakanae or foolish seedling disease caused by Fusa-rium fujikuroi Nirenberg (teleomorph: Gibberella fujikuroi), is a destructive seed-borne and seed-transmitted disease. It was first identified in 1828 in Japan and now is widely distri-buted throughout rice growing countries [17]. Its incidence rate was 3-80% in major rice growing countries, in-cluding China, India, Bangladesh, and Korea. Yield losses from bakanae disease were estimated at about 20-50% in Japan and 15-25% in India [15]. The typical symptoms of the disease are slender and abnormally elongated seedlings in response to gibberellins produced by the fungal pathogen[17]. In severe cases, seed sterility and dried dead seedlings were found to be frequently in heavily infected seeds.

The bakanae disease of rice was first discovered in some areas of Korea in the 1960s, but the incidence rate of the disease was significantly low (less than 3%) until the early 2000s due to effective seed disinfection with chemical fungicides, such as benomyl and prochloraz [5, 14, 18]. However, the occurrence of the disease increased again, resulting in a higher incidence rate of 28.8% in 2006. This may be linked to climate change or global warming which creates optimal conditions for fungal growth during the rice nursing and ripening stage [3]. Furthermore, the occurrence of fungicide-resistant fungal strains is one of the reasons for failure in disease control [5,14], thus it is critical to develop effective and safe materials for the disease management.

Hypochlorous acid (HOCl) is a typical chlorine-based dis-infectant produced by myeloperoxidase-mediated perox-idation of chloride ions in activated immune cells to prevent microbial infections [1]. It showed a strong and broad spec-trum of microbicidal activity against bacteria, fungi, and viruses. Although the precise mechanism underlying the microbicidal activity of HOCl has not yet been elucidated, its possible action is thought to be involved in inhibition of glucose metabolism, depletion of adenine nucleotides, inhibition of DNA synthesis, and protein unfolding and aggregation[7,19]. Otherwise, HOCl showed non-irritating, non-sensitizing, and low cytotoxicity to mammalian cells in the range of an effective microbicidal concentration. Thus, HOCl has been approved by the FDA, USDA, and EPA for uses as a disinfectant, food additive, or sanitizer in various fields of medical, agriculture, and food industry [2, 7, 20].

In chlorine-based disinfectants, free available chlorine(FAC) mainly exists in three different forms, Cl₂, HOCl, and OCl⁻ [2,7]. HOCl is a weak acid (pKa=7.5, 25℃) that dissociates readily to H⁺ and OCl^- depending on the pH in aqueous solutions. In the pH range of 5.0 to 6.5, HOCl is the predominant species (>95%) in the solution, and its dissociation is minimal. This solution is often called slightly acidic hypochlorous acid water (SAHW). In a solution above pH 9.5, the composition of OCl^- nearly reaches the 100% level. A typical strong alkaline solution composed of OClis sodium hypochlorite (NaOCl). At pH values less than 3.5, the solution exists as a mixture of aqueous Cl₂, Cl₂ gas, trichloride (Cl₃⁻), and HOCl.

In this study, we investigated the disease control activity of SAHW against the bakanae of rice caused by F. fujikuroi, and its effectiveness was demonstrated.

Materials and Methods

Materials

Sixteen F. fujikuroi strains composed of sensitive and resistant strains to chemical fungicides, prochloraz and benomyl, were obtained from the Korean Agricultural Culture Collection (KACC, Jeonju, Korea) and incubated at 27℃ on potato dextrose agar (PDA). A fungal spore of each F. fujikuroi strain was prepared from aerial mycelium in 5 ml of sterile water by gently scraping the surface with a spatula. The spore suspension was passed through 3 layers of cheesecloth to remove the debris and adjust the density to 1x10⁶ spore/ml. SAHW (HOCLer, Cosmic Round Korea Co., Ltd.) prepared by electrolysis of diluted HCl in a chamber of a non-membrane electrolytic cells was used in this experiment. The pH range of SAHW is 5 to 6.5, and the free available chlorine (FAC) concentration is 20±10 ppm.

Antifungal activity of SAHW

The antifungal activity of SAHW was analyzed by the microplate growth inhibition assay. Briefly, the fungal spores isolated from 16 F. fujikuroi strains were inoculated in PDB to a concentration of 2×10⁴ spore/ml. A total of 50 μl of PDB medium containing spores was mixed with 50 μl of various concentrations of SAHW (typically 2-fold dilutions from 1.25±0.6 to 20±10 ppm FAC) in a 96-well microplate. After incubation at 27℃ for 24-36 hr spore germination and hyphal growth was determined by measurement of the absorbance at 595 nm with a microplate reader (EnSpire, Perkin Elmer, USA). Sterile water was used as a control. Minimum inhibitory concentrations required to inhibit the growth of 50 and 90% (MIC₅₀ and MIC₉₀) were determined from dose-response curves.

Fungicidal activity of SAHW

To understand the molecular mechanism underlying the microbicidal action of SAHW, a viable count using a colony-forming unit (CFU) assay and scanning electron microscope (SEM) analysis was conducted. Fungal spores incubated in PDB (2×10⁴ spore/ml) for 12 hr at 27℃ were treated with an equal volume of SAHW (10±5 ppm FAC) for 1 and 5 min. As a control, fungal spores were treated with sterile water for 5 min. Samples were then serially diluted with sterile water and plated on PDA medium. CFU was determined after 2-3 days of incubation at 27℃. Three replicates were used for each treatment. SEM analysis was carried out as standard protocol. Briefly, PDB medium containing fungal spores (fungicide-resistant KACC44004 and fungicide-sensitive KACC44018) was dropped on poly-L-lysine-coated glass coverslips (Corning, USA) in a 24-well microplate. After 12 hr of incubation at 27℃, the coverslips were incubated with SAHW (10±5 ppm FAC) for 5 min at room temperature. Then, samples were fixed and sequentially dehydrated from 50 to 100% ethanol. The fungal morphology was visualized in a Supra 40VP FE-SEM (Carl Zeiss, Germany).

Seed disinfection and biocontrol activity of SAHW

To investigate the effect of seed disinfection and biocontrol activity of SAHW, the rice seeds (Oryza sativa L. cv.Joonam) were artificially inoculated with spores of F. fujikuroi strains (KACC44004 and KACC44018). Briefly, dry seeds were surface sterilized by dipping in 70% ethanol for 1 minute followed by thoroughly rinsing in sterile water. Then, the seeds were dipped in water containing fungal spores(1×10⁴ spore/ml) for 6 hr at 27℃ with gentle agitation. After inoculation, the seeds were thoroughly rinsed with sterile water and treated with SAHW containing 5±2.5, 10±5 and 20±10 ppm FAC for 1 hr with gentle agitation. The intact and healthy seeds were then planted on a half-strength MS agar medium (Duchefa Biochemie, Netherlands). To investigate the effect of the chemical durability on seed disinfection, the inoculated seeds were treated with SAHW containing 20±10 ppm FAC for 12 hr in three different ways: I, single treatment for 12 hr; II, a treatment for 3 hr, drain and re-treatment with a fresh solution for 6 hr (two times); III, treatments with a fresh solution every 3 hr (four times). The seeds were then planted on a half-strength MS agar medium or in commercial nursery potting soil (Nongwoo Bio Co., Korea). After 10 days and 20 days of incubation in a growth chamber at 30℃, seed disinfection and disease incidence were evaluated, respectively.

Molecular identification of F. fujikuroi by polymerase chain reaction (PCR)

To identify whether the microorganism contaminated on rice seeds and seedlings was F. fujikuroi, PCR analysis was carried out using the species-specific primer. Contaminated seed or seedling tissue (5×5 mm) was incubated in 100 ml PDB medium for 2 days at 27℃. After the sample was ground into a powder in liquid N₂, total DNA was extracted using a Plant/Fungi DNA Isolation Kit (Sigma, USA) according to the manufacturer’s manuals. PCR was performed in a total volume of 20 μl containing 50 ng of DNA and 10 ng of an F. fujikuroi-specific primer set [4], BFspF (5'-TGAGA CTTGTGTCTGAGAGCT-3') and BFspR (5'-GATAACATC ATAACTCCTGCG-3'). The PCR was performed with following program: denaturation at 94℃ for 3 minutes and 30 cycles of 94℃ for 30 seconds, 55°℃ for 30 seconds and 72℃ for 1 min. The 547 bp of PCR product was analyzed by electrophoresis in 1.5% agarose gel.

Statistical analysis

Data are represented as the mean ± standard error (SE). The values between experiments were analyzed by Tukey’s multiple comparison test at p<0.05. Analysis of variance(ANOVA) was carried out by OriginPro software (OriginLab Co., USA).

Results

Antifungal activity of SAHW against F. fujikuroi strains

SAHW (pH 5.0-6.5, FAC of 20±10 ppm) prepared by electrolysis of diluted HCl in a chamber of a non-membrane electrolytic cell was used in an antifungal assay of F. fujikuroi causing bakanae disease of rice. Sixteen strains of F. fujikuroi were obtained from KACC (Table 1). Some of them were resistant strains to chemical fungicides used in seed disinfection, such as prochloraz and benomyl.

Table 1. Antifungal activities of SAHW on 16 strains of Fusarium fujikuroi Nirenberg isolated from rice in Korea

^aAntifungal activities of SAHW were carried out by a microplate growth inhibition assay.

^b,cMIC₅₀ and MIC90 were determined from dose-response growth curves. The concentration of FAC in SAHW is represented as ppm. Static analysis indicates no significant differences between the values in the same column by Tukey’s multiple comparison test with p<0.05.

In the microplate-based growth inhibition assay, SAHW showed strong inhibitory activities on spore germination of F. fujikuroi, irrespective of chemical fungicide-resistant and sensitive strains (Table 1). The MIC₅₀ values of FAC in SAHW for all examined strains ranged from 2.22±1.11 to 2.62±1.31 ppm, and MIC90 values were 5±2.5 ppm without a significant difference among the strains (n=3; p>0.05).

Fungicidal activity of SAHW

To investigate the mode of action, the viability of F. fujikuroi cells was analyzed by a CFU assay after treatment with an inhibitory concentration of SAHW for 1 and 5 min. From the experiments using 16 strains of F. fujikuroi, representative data for the sensitive strain (KACC44004) and resistant strain(KACC44018) are shown in Fig. 1A. In comparison with the water-treated control, treatment of hyphal growing cells with SAHW containing 10±5 ppm FAC induced rapid cell death in a majority of cells (97.1-99.9%) within 1 min, and the viability was completely lost after 5 min with no significant difference among the strains (n=3; p>0.05). To further understand the effect of SAHW on fungicidal activity, the surface morphologies of F. fujikuroi cells were analyzed by SEM (Fig. 1B). After treatment with SAHW containing 10±5 ppm FAC for 5 min, the majority of cells showed the leakage of cytoplasmic contents, small or large aggregates on the cell surface and lethal damage on hyphae in both the KACC44004 and KACC44018 strains. Furthermore, approximately 50% of cells contained small or large pores on the cell wall. However, no visible differences in morphologies were observed in the majority of cells when the cells were treated with water. The morphological changes were consistent with a previous observation in which SAHW induced protein aggregation and pore-formation on the cell surface through membrane leakage [2,7]. These results suggest that the primary action of SAHW is leading to cellular leakage, which in turn causes disruption of cell integrity and cell death. Many chemical fungicides targeted specific biomolecules or the metabolic pathway to exert the microbicidal action. Therefore, SAHW might gain attention as an attractive microbicidal agent that minimizes the occurrences of resistant strains due to its general action.

Fig. 1. Effect of SAHW on cell viability and morphology of F. fujikuroi cells. (A) Hyphal growing cells of F. fujikuroi strains (KACC44004 and KACC44018) were treated with water as control or SAHW containing 10±5 ppm FAC for 1 and 5 min. CFU were determined after incubation for 2-3 days at 27℃ on PDA medium. Error bars represent the standard error of the mean (n=3). Different letters (a-b) indicate statistically significant differences(p<0.05). (B) Hyphal growing cells treated with water or SAHW containing 10±5 ppm FAC for 5 min were fixed and processed for SEM analysis. Representative images are shown. Scale bar, 2 μm.

Seed disinfection and management of bakanae disease of rice by SAHW

To investigate the effect of SAHW on seed disinfection of rice, artificially inoculated seeds with F. fujikuroi strains(KACC44004 and KACC44018) were immersed in various concentrations of SAHW for 1 hr. Then, the seeds were aseptically incubated on MS agar medium, and the rate of seed disinfection was determined after 10 days of incubation (Fig. 2A). When the inoculated seeds were immersed in water as a control, all seeds showed the growth of fungi on their surface following the incubation. Furthermore, the hyphal growth of fungi leads to the failure of seed germination and seedling growth (Fig. 2C). PCR amplification with species-specific primers and sequencing analysis of its products revealed that the fungi grown on the seeds were F. fujikuroi(Fig. 2D). Otherwise, the inoculated seeds immersed in SAHW showed a remarkably decreased and delayed appearance of fungal growth on the seeds in a dose-dependent manner. The immersion in SAHW containing 5±2.5 and 10±5 ppm FAC was equally effective for up to 3 days of incubation, but thereafter fungal hyphae developed on 74-83% and 38~41% of seeds, respectively (Fig. 2A). However, when the seeds were immersed in SAHW containing 20±10 ppm FAC, the emergence rate of fungal growth was 17-21% during the incubation period. Furthermore, the seeds produced normal plant growth and development without adverse effects (Fig. 2C).

 

Fig. 2. Seed disinfection by treatment with SAHW.

(A) Seed disinfection depending on the concentration of SAHW. Rice seeds inoculated by F. fujikuroi strains (KACC44004 and KACC44018) were treated with water as control or SAHW containing 5±2.5, 10±5 and 20±10 ppm FAC for 1 hr. Different letters (a-d) indicate statistically significant differences (p<0.05). (B) Effect of exchange of SAHW with fresh solution during 12 hr of treatment. The inoculated seeds were treated with water or SAHW containing 20±10 ppm FAC with three different methods: (I) treatment with SAHW for 12 hr, (II) treatment with SAHW for 3 hr, rinsed and re-treatment with fresh solution for 9 h, and (III) treatment with SAHW every 3 hr with fresh solution during the incubation period. The seeds were then planted on a half-strength MS agar media. After 10 days of incubation, the rate of seeds with fungal growth was determined. Error bars represent the standard error of the mean. Different letters (a-c) indicate statistically significant differences (p<0.05). (C) Typical images of seed disinfection from (B) seeds treated by method III. Scale bar, 2 cm. (D) PCR amplification with species-specific primers. Total DNA was isolated from rice seeds treated with SAHW or water control as shown in (C) and F. fujikuroi. It was noted that the 547 bp of amplified PCR products was derived from F. fujikuroi.

The effectiveness of seed disinfection depends on the concentration and exposure time of the disinfectant. When the inoculated seeds were immersed in SAHW containing 20±10ppm FAC for a longer time up to 12 hr, no significant improvement in seed disinfection was observed (data not shown). It was reported that HOCl is chemically unstable against ultraviolet light, contact with air by agitation, elevated temperature and the presence of organic compounds[6,10]. It might be possible that HOCl in SAHW was rapidly consumed by an oxidation reaction and significantly decreased its microbicidal activity during seed disinfection. To validate this possibility, we tried to change SAHW more than once with a fresh solution during the 12 hr of the incubation period (Fig. 2B). When SAHW was exchanged three times with a fresh solution during the incubation period, the effectiveness of seed disinfection was significantly improved by up to 95–98%, compared to single and double treatments (n=3; p<0.05). These results suggest that the chemical stability of SAHW should be considered for effective disinfection when it practically applies to seed disinfection.

To examine the effect of SAHW on the management of bakanae disease, the disinfected seeds were planted in a soil pot and the incidence of disease symptoms was monitored after 20 days of cultivation (Table 2). In the water control, a majority of seeds produced severe disease symptoms including failure of seed germination, seedling death during growth and seedlings with dense hyphal growth due to heavy infection of F. fujikuroi. In contrast, the development of bakanae symptoms was significantly suppressed by treatment with SAHW. Although some seedlings showed typical bakanae disease symptoms including abnormal elongation with pale green flag leaves, the control efficiencies of SAHW containing 10±5 and 20±10 ppm FAC were 73.7~76.9% and 90.1~92.6%, respectively. 

Table 2. Control effect of SAHW on bakanae disease caused by F. fujikuroi (KACC44004 and KACC44018)

^a-dDifferent letters within the column indicate statistically significant differences by Tukey’s multiple comparison test (p<0.05). 

* Infected seeds were treated with fresh SAHW or water (control) every 3 hr for a total 12 hr. 

**Not determined due to severe symptoms including failure of seed germination, seedling death and rotted seedlings.

Discussion

SAHW showed strong microbicidal activities against F.fujikuroi, irrespective of chemical fungicide-resistant and sensitive strains (Table 1). These activities were similar or higher levels, compared to those against plant pathogens including Colletotrichum acutatum, Phytophthora capsici, Botrytis cinerea with MIC90 values of 6-15 ppm [20], and much higher than those against clinical isolates of bacteria, mold and yeast with an MIC90 value of 30 ppm [2,8]. Moreover, it was reported that SAHW is effective against Staphylococcus aureus strains both susceptible and resistant to the antibiotic methicillin [10]. On the contrary, a few studies have suggested that an HOCl solution possesses higher bacteriocidal and virucidal activities than fungicidal properties [2]. Nevertheless, our results suggested that SAHW may be a good compound for the control of F. fujikuroi strains including those showing resistance to chemical fungicides, at least prochloraz and benomyl.

Although the mode of action of HOCl solution is not yet fully understood, many studies reported that the un-dissociated form of HOCl is the primary factor affecting microbicidal activity than the negative-charged membrane-impermeable form of OCl^- or molecular Cl₂ within its effective antimicrobial concentration range [2,7]. They have also suggested that the non-charged HOCl is able to penetrate the cell membranes, which in turn exerts its microbicidal activity through the irreversible oxidation of thiol and amino groups in key metabolic molecules including proteins, nucleotides and lipids [19]. However, it was also noted that a high oxidation-reduction potential (ORP) contributes to bactericidal activity, in combination with pH (a high H⁺ concentration) and FAC in the solution. Thus, the precise mechanism underlying the action of SAHW in the cell death process requires further investigation. 

Among the chlorine-based disinfectants, SAHW is the most desirable disinfectant for several reasons. First, unlike strong acidic HOCl or alkaline OCl^- solutions, the pH range of SAHW is a more suitable for the environment, plant growth and human health. Second, it does not release poisonous Cl₂ gas like strong acidic HOCl solutions. Third, when it combines with acids or organic materials, toxic fumes or harmful by-products, such as trihalomethane (THM), are not produced compared to alkaline OCl^- solutions. Thus, it is more suitable to use in enclosed space, such as green house. Fourth, SAHW is 80-100 times more effective as a disinfectant agent than an equivalent concentration of an OCl^- solution [6]. Thus, SAHW has been widely used in the medical, agriculture, and food industry fields [2, 7, 20].

Seed disinfection to control the rice bakanae disease has been carried out using several different methods, including treatment with various chemical fungicides, biocontrol using an endophytic Bacillus [9], treatment with seed coat extracts of Ginkgo biloba [16] and immersion in hot water [18]. However, they were somewhat ineffective on severely infected rice seeds or strains resistant to chemical fungicides[5, 14, 18]. In this study, we demonstrated the effectiveness of SAHW as a potential alternative to fungicide in the control of rice bakanae diseases.