In vivo Testing of Plant Extracts in Controlling Rice Brown Spot Disease Through Folia Application

Rice is one of the most important cereal crops in Thailand, planted for both local consumption and exports. In 2014, Thailand was the world’s number one exporter of milled rice (Charoenrak and Chamswarng, 2015). Unfortunately, rice production confronts many diseases and infections, including fungi, which constrain both quantitative and qualitative yield losses of up to 45% (Nalley et al., 2016; Law et al., 2017; Raveloson et al., 2018). Rice brown spot disease caused by Bipolaris oryzae (Breda de Haan) Shoemaker [telemorph: Cochliobolus miyabeanus (Ito and Kuribayashi) Dreches. ex Dastur] was recorded as one of the most significant diseases in rice and results in yield losses of about 10% whenever the disease occurs and up to 90% in grain yield losses (Ghose 2000; Savary et al., 2000; Sunder et al., 2014). The disease is widespread worldwide in rice-growing areas and it manifests through blemishes that are visible on several parts of the rice plant, including the leaves, sheath, panicles and grain (Mohd Anuar et al., 2020). The typical brown spot symptom appears as a brown oval or cylindrical spot with a gray or whitish center surrounded by a yellow halo on rice leaves in the growing stage until the maturity stage (Sunder et al., 2014; Kumari et al., 2015). However, grain discoloration occurs after B. oryzae infects the rice glumes (Marchetti and Petersen, 1984).
 
While the application of synthetic fungicides was mainly used for rice disease control, the repeated and continued use of synthetic fungicides has resulted in the development of pathogen resistance and harm to the environment, the local ecology, non-target microorganisms and human health (Thind, 2012; Nicolopoulou-Stamati et al., 2016; Van de Wouw et al., 2017). Hence, there is an urgent need to solve these problems and find an effective way to manage these rice diseases, replace the use of synthetic fungicides and promote organic crop production and sustainable agriculture. Nowadays, several studies seek to discover new potential antifungal activity from biological control agents and plant extracts as botanical fungicides to develop an alternative bio-fungicide to control rice disease with effectiveness similar to that of synthetic fungicides (Zhang et al., 2014). Some plant species may contain rich amounts of bioactive compounds by producing diverse groups of secondary metabolites against plant pathogens (Naveenkumar et al., 2017; Dethoup et al., 2019; Kokkrua et al., 2020). Jantasorn et al., (2016) reported antifungal activity from the extracts of Hydnocarpus anthelminthicus and Xanthophyllum lanceatum, best effective against Pyricularia oryzae and Rhizoctonia solani, which cause rice diseases in vitro. However, the H. anthelminthicus leaves extract showed a strong inhibitory effect on the appressorium formation and conidial germination of Colletotrichum higginsianum, causing anthracnose disease in Chinese cabbage (Hsieh, 2018). The crude ethanol extract of Acorus calamus at a concentration of 5 g/L displayed the ability to significantly reduce brown spot incidence by 47% (Dethoup et al., 2019). Indeed, the use of natural products to develop botanical fungicides for fungal management is considered one of the better alternatives that can decrease the current use of synthetic fungicides, which contaminates and poisons other organisms present in the environment (Gujar et al., 2012; Plodpai et al., 2013; Shetty and Shruthi, 2015). Therefore, the objective of this study was to investigate the antifungal effects of the plant extracts of H. anthelminthicus, C. magna, C. sappan, X. lanceatum and C. brachiata on B. oryzae in vitro as well as their fungal activity to control the brown spot disease of rice seedlings, as assessed under greenhouse conditions.

Plant materials and extractions
 
Five plant samples-namely, H. anthelminthicus (fruit), C. magna (fruit), C. sappan (bark), X. lanceatum (fruit) and C. brachiata (bark)-were collected from the riparian forest at Bodhivijjalaya College, Srinakharinwirot University, Sakaeo campus. Rice cv. KDML 105 seeds were obtained from the Surin Rice Research Center in Surin Province, Thailand. Each plant material was washed with tap water, air-dried at room temperature, cut into small pieces, ground into a fine powder and stored at 4°C until used. The extraction method was then performed (Jantasorn et al., 2016; Jantasorn et al., 2017). Briefly, 200 g of each plant powder sample was extracted twice, macerated with 95% ethanol and incubated at room temperature for 1 week. The aqueous ethanol extracts were filtered through three-layer sterile cheesecloth and then the solutions were concentrated under reduced pressure to produce the crude ethanol extracts.
 
In vitro mycelial growth inhibition test

The mycelial growth inhibition activity of the plant crude extracts was evaluated on B. oryzae in vitro via the dilution plate method, as described by (Dethoup et al., 2019). Briefly, each plant crude extract was dissolved in dimethyl sulfoxide (DMSO) and diluted in sterile water using the serial dilution method. Then 1 mL of each crude extract solution was mixed with 9 mL of warm potato dextrose agar (PDA) to obtain the final concentrations of 1, 10, 100, 1,000 and 10,000 ppm in separate petri dishes. A mycelial plug (5 mm in diameter) of B. oryzae obtained from an active growing colony of a seven-day-old culture was placed at the center of each petri dish. A mycelial plug control for tested fungus was placed on the petri dishes containing 1% DMSO. Each tested treatment plate was incubated at room temperature (28±3°C) for 14 days and the experiment was repeated twice with five replicates. Mycelial growth inhibition (MGI) was calculated using the following formula:
 

%MGI = [(G1−G2)/G1] × 100

Where
G1 represents the diameter of the colony radius of the fungi in the negative control and G2 is the diameter of the colony radius of B. oryzae treated with a plant crude extract at different concentrations.
 
Evaluation of plant crude extracts against brown spot disease on rice seedlings under greenhouse conditions
 
Rice cv. KDML 105 was used in this study, which is susceptible to rice brown spot disease caused by B. oryzae. The rice seeds were surface disinfected with a 1% (v/v) sodium hypochlorite solution (Dethoup et al., 2019). Then the rice seeds were planted in a plastic tray and kept in nurseries for 7 days. Five seedlings of the same height and vigor were transplanted to a plastic pot (18 cm in diameter, 12 cm in height) containing sterile clay soil, placed in a greenhouse at 28±2°C for 4 weeks and watered as needed to maintain a water level of 1 cm above the soil surface throughout the experiment. The experiments were repeated twice and the pot used in a completely randomized design with five replicates. Five plant crude extracts were dissolved separately in DMSO and diluted in sterile water to give final concentrations of 10,000 and 50,000 ppm. The spore suspensions containing 106 spores mL-1 of B. oryzae were prepared as described by (Komhorm et al., 2021). The effects of the five plant crude extracts were evaluated to suppress rice brown spot disease under greenhouse conditions in rice aged 30 days and 45 days. The rice seedlings were sprayed once 30 days after transplanting (DAT) and again at 45 DAT with 30 mL of each concentration of crude extract containing 0.01% (v/v) Tween-20 per pot on both sides of the leaves 24 hours before B. oryzae inoculation. Then 30 mL spore suspensions (106 spores mL-1) of B. oryzae containing 0.01% (v/v) Tween-20 were inoculated on the treated rice seedling in each of the five pots (replications) per treatment; the treated pots were incubated at 28±2°C and 90%-100% humidity for 1 day and placed in the greenhouse. The five pots of rice seedlings were treated with 1% DMSO containing 0.01% (v/v) Tween-20 as a negative control. Disease incidence was evaluated 7 days after pathogen inoculation. Three leaves were collected from the top part of each plant and inspected for disease incidence (Komhorm et al., 2021). Difenoconazole (25% W/V EC, Syngenta Crop Science Co. Ltd.) was applied as a positive treatment.
 
Statistical analysis
 
The data was analyzed using the statistical program Statistix8 (Analytical Software, SXW, Tallahassee, FL, USA). The data in the experiments was evaluated using analysis of variance (ANOVA); the means were compared using least significance difference (LSD) with 95% statistical significance (p<0.05). Therefore, the data was obtained from the repeated experiments, pooled and submitted to analysis.

In vitro antifungal activity of plant crude extracts against B. oryzae
 
The antifungal activity levels of the H. anthelminthicus, C. magna, C. sappan, X. lanceatum and C. brachiata crude extracts were tested against the mycelial growth of B. oryzae at different concentrations (1, 10, 100, 1,000 and 10,000 ppm) in in vitro conditions. The growth of B. oryzae was found to have an inverse relationship with the concentration of the crude extracts. The H. anthelminthicus crude extract showed the best antifungal activity against B. oryzae at the highest dose tested, causing 93% mycelial growth inhibition. Additionally, the X. lanceatum and C. brachiata extracts at a concentration of 10,000 ppm exhibited significant mycelial growth inhibition of B. oryzae, causing 82% and 67% inhibition compared with the control treatment (Fig 1). Meanwhile, the C. magna and C. sappan extracts inhibited the mycelial growth of B. oryzae via the dilution plate method on PDA by more than 50% (data not showed) and at low concentrations (1, 10 and 100 ppm), each crude extract failed to inhibit the mycelial growth of the brown spot disease pathogen.


 
Effect of five plant crude extracts against B. oryzae affecting brown spot disease in rice cv. KDML 105 under greenhouse conditions
 
The antifungal activity test results of the five plant crude extracts at two concentrations in controlling B. oryzae causing rice brown spot disease compared with the control in greenhouse conditions are shown in Fig 2 and 3. H. anthelminthicus, X. lanceatum, C. brachiata, C. magna and C. sappan effectively suppressed the incidence of rice brown spot disease caused by B. oryzae when applied once at 50,000 ppm (30 DAT) compared with the water control (Fig 2). However, when the rice seedlings were sprayed twice, once 30 DAT and again 45 DAT, with the H. anthelminthicus, X. lanceatum, C. brachiata, C. magna and C. sappan crude extracts at a concentration of 10,000 ppm, they displayed the best levels of reduction and suppression of the development of brown spot incidence (Fig 3). Interestingly, the H. anthelminthicus crude extract at 10,000 ppm was found to induce the greatest level of suppression in the development of brown spot symptoms on rice seedlings when the plants were treated twice, 30 DAT and 45 DAT (Fig 4). However, the efficacy of the H. anthelminthicus crude extract being applied twice displayed the best reduction in the percentage of disease incidence of rice brown spot disease in greenhouse conditions, similar to the positive control with synthetic fungicides (difenoconazole). Furthermore, the rice seedlings in the water treatment showed typical brown spot symptoms.

​

​

​

Nowadays, several researchers seek new approaches to decrease the use of synthetic fungicides in rice disease management, which causes pathogen resistance. However, rice diseases caused by fungi can constrain both quality and yield losses of more than 50% in rice production (Spence et al., 2014; Awla et al., 2017). Biological control and botanical fungicides are promising approaches to rice disease management; however, the bioactive compounds from plant extracts to control rice diseases are still limited (Torres et al., 2017; Shao et al., 2018). In this study, the main antifungal activity of the H. anthelminthicus extract was observed both in vitro and in vivo. The H. anthelminthicus extract displayed potent in vitro antifungal activity against B. oryzae at high doses. Moreover, the antifungal activity of the H. anthelminthicus extract inhibited the mycelial growth of Pyricularia oryzae, Rhizoctonia solani and Phytophthora palmivora by 100% at a concentration of 10,000 ppm and the X. lanceatum extract at high doses showed a complete mycelial growth inhibition of P. oryzae (Jantasorn et al., 2016). Hsieh (2018) reported that the H. anthelminthicus crude ethanol extract from leaves at a concentration of 2.5% (v/v) displayed a strong inhibitory effect on the conidia germination and appressorium formation of Colletotrichum higginsianum, causing anthracnose in Chinese cabbage. However, several studies reported that the efficacy levels of plant extracts against B. oryzae have been tested (Bhuyan et al., 2010; Nguefack et al., 2013; Dethoup et al., 2019; Kokkrua et al., 2020).

In greenhouse testing, the plant extracts had different levels of inhibitory activity to suppress the development of brown spot symptoms on rice seedlings and significantly reduced the incidence of the disease. In addition, the concentration and spraying quantity of the H. anthelminthicus, X. lanceatum, C. brachiata, C. magna and C. sappan crude extracts are critical for the effective control of rice brown spot disease. The X. lanceatum extract at a concentration of 50,000 ppm displayed the best effects in suppressing the development of brown spot symptoms caused by B. oryzae when applied once 30 DAT before pathogen inoculation. However, the H. anthelminthicus extract, when applied once at both concentrations (10,000 and 50,000 ppm) 30 DAT, showed a strong antifungal effect on B. oryzae growth. In addition, the efficacy of H. anthelminthicus against B. oryzae when applied once and twice at 10,000 ppm at both stages of growth (30 DAT and 45 DAT) was tested under greenhouse conditions. This phenomenon may be due to differences in the fungicidal activity of plant extracts. The percentage of brown spot disease incidence was lower than in the plants treated with the X. lanceatum, C. brachiata, C. magna and C. sappan extracts. This revealed that the H. anthelminthicus extract had higher fungicidal activity compared with the other plant extracts tested in this study. Thus, the H. anthelminthicus extract exerts the highest level of preventive activity to reduce the disease incidence of B. oryzae under greenhouse conditions. Also, the efficacy of the H. anthelminthicus ethanol crude extract at a concentration of 0.5% significantly reduced the incidence and severity of anthracnose in Chinese cabbage under greenhouse conditions (Hsieh, 2018).

Rice brown spot disease caused by B. oryzae can constrain both the yield and quality of rice production; many attempts have been made to find plant extracts to control this disease. Although many have reported on the antifungal activity of plant extracts against B. oryzae in vitro and in vivo (Nguefack et al., 2013; Dethoup et al., 2018; Dethoup et al., 2019), none have reported on the effect of the H. anthelminthicus crude extract against this disease in vivo. Our results in the greenhouse experiment indicated that the spraying of plant crude extracts once and twice on the leaves of the rice seedling plants resulted in a significant decrease in pathogen infection by B. oryzae, whereby all the treatments with the tested plant extracts reduced the disease incidence and suppressed the development of brown spot symptoms in rice seedlings compared with the unprotected control. Based on our results, the H. anthelminthicus crude extract at a concentration of 10,000 ppm was the most effective in suppressing the disease incidence by more than 80%, with two sprays (30 DAT and 45 DAT) and inoculation with B. oryzae. In this study, the rice seedling leaves did not show any phytotoxic symptoms when the H. anthelminthicus crude extract was applied at the highest dose of 50,000 ppm. So far, botanical fungicide products derived from plant extracts are still limited to development for commercialization. This study indicated that the H. anthelminthicus crude extract has higher antifungal activity against B. oryzae, which causes brown spot disease in rice seedlings, since it caused a reduction in disease incidence at 10,000 ppm both in vitro and in vivo. The H. anthelminthicus crude extract is safer for human beings and the results of this study demonstrate that this crude extract is a promising alternative botanical fungicide against B. oryzae to replace the use of synthetic fungicides.

Our results demonstrated that the H. anthelminthicus crude extract exhibited significant and potent botanical fungicide activity against B. oryzae, preventing it from causing the foliar disease of rice in vitro and in vivo. It was shown to strongly prevent B. oryzae infection and reduce disease development. Moreover, the H. anthelminthicus crude extract displayed the most effective antifungal activity against rice brown spot disease and reduced the incidence of this disease. Thus, the H. anthelminthicus crude extract can be applied to control rice brown spot disease caused by B. oryzae and can be developed as an alternative botanical fungicide to reduce the use of chemical fungicides. However, further studies are required to test the H. anthelminthicus crude extract against B. oryzae and to evaluate its control efficiency compared with that of synthetic fungicides in paddy fields.

This research was financially supported by the Strategic Wisdom and Research Institute, Srinakharinwirot University, under the project “Efficacy of plant crude extract against brown spot disease of rice caused by Bipolaris oryzae under greenhouse condition (project no. 105/2563)”.

The author declare that they have no conflict of interest.

This article does not contain any studies involving human participants or animals by any of the authors.