Inhibitory Effects of Bamboo Leaf on the Growth of Pyricularia grisea Fungus

In this study, the effects of bamboo leaf were examined on mycelial growth of Pyricularia grisea, a fungus of rice blast disease that causes a great loss in rice production. The hexane extract exhibited maximal reduction on growth of P. grisea (IC50 = 0.62 mg/mL), followed by aqueous and ethyl acetate extracts, while the methanol extract was least effective (IC50 = 9.71 mg/mL). At 0.5–1.0 mg/mL doses, all extracting solvents showed inhibition on the growth of P. grisea, but at a 0.1 mg/mL concentration, the antifungal activity was solely observed on hexane and ethyl acetate extracts. By GC-MS (gas chromatography-mass spectrometry), 25 constituents were identified, principally belonging to long-chain fatty acids, sterols, phenols, phenolic acids, volatile oils, and derivatives of terpenes. It was suggested that compounds originated from hexane and ethyl acetate extracts such as fatty acids, oils, and phenols, and their derivatives were responsible for the antifungal activity of bamboo leaf. Non-polar constituents were accountable for the antifungal activity, although water-soluble compounds may play a role. Bamboo leaf appears to be a potent natural source to manage the infestation of P. grisea in rice cultivation.


Introduction
Fungal diseases have caused extensive yield loss in many crops and thus have become a major challenge for agricultural production [1].Rice blast is caused by a highly variable fungal pathogen, Pyricularia grisea.It is a serious disease, affecting rice yield with up to 70-80% losses [2].The use of rice varieties resistant to this fungus has been shown to be effective.However, the resistance levels governed by the resistance genes have been reported to be inherently unstable and fast-evolving [3,4].On the other hand, the application of synthetic chemical fungicides has certain advantages, but they also apparently result in environmental and health problems.In recent years, the use of natural products as environmentally friendly agricultural chemicals to manage plant diseases, including the biological control on P. grisea, has been targeted to a greater extent.
It was documented that the crude extract of Epicoccum sp., the leaf extracts of Prosophis juliflora, and Ziziphus sp inhibited the mycelial growth of P. grisea [5,6].In addition, extracts from E. aromatica, P. guineense, and G. kola could serve as bio-fungicides against the growth of P. grisea [7].Several other plants have been tested and showed significant results against P. grisea [7][8][9].However, the application of plant extracts in agricultural practice is labor-intensive and costly.Therefore, the search for bioactive chemicals from plants against the fungus P. grisea is much desired.
Bamboo grows abundantly in the subtropics and tropics.It belongs to the family Poacaea (grass), sub-family Bambusoideae, and tribe Bambuseae.Phyllostachys pubescens is the major species of bamboo in Japan and is widely distributed through the country [10].Bamboo might be made up of rich phytochemicals, which should be investigated and exploited [11].Gong et al. [12] reported that the 1-butanol fraction of bamboo leaf possessed strong antioxidant activity and high exhibition of total phenol and flavonoid contents, and various phenolic acids.Moreover, 1-butanol, ethyl acetate, and water fractions of bamboo extracts expressed a high capacity for anti-oxidation.Caffeic acid was the principal compound that exerted the highest antioxidant capacity on DPPH (2,2-diphenyl-1-picrylhydrazyl) and FRAP (Ferric-Reducing Antioxidant Power) assays [12].Similarly, polysaccharides extracted from Moso bamboo-shoot [13] and bamboo leaf [14] also showed strong significant antioxidant activity.In addition, the leaf and root extracts of bamboo exerted inhibitory effects on growth of groundnut and corn seedlings [15,16].The pharmacological properties, including anti-inflammatory, anticancer, and antibacterial activities of bamboo have been described [12,17].From this evidence, it is proposed that bamboo leaf may possess potent antifungal activity and may be successfully applied in the management of plant pathogens.This study was therefore carried out to evaluate whether bamboo leaf can be utilized for the biological control of P. grisea, a problematic fungus in rice production.The analysis of potent phytochemicals in bamboo leaf was also conducted.

Collection and Preparation of Bamboo Leaf Extract
The leaves were collected from different bamboo trees grown near Higashi Hiroshima campus of Hiroshima University, Japan in September 2016, and air-dried for two weeks.The leaf powder (200 g) was made by a grinder and immersed to methanol for three days at room temperature (23-25 • C).After filtration and vacuum evaporation, the precipitate was successively partitioned into solvents with different polarities, including hexane, ethyl acetate, 1-butanol, and water.The obtained extracts were separately evaporated and diluted to different concentrations for further tests and analyses.

GC-MS Analysis
Each extract, obtained as described above, was evaporated, filtered, and diluted in acetone at 10 ppm for analysis by a GC-MS instrument (JMS-T100 GCV, JEOL Ltd., Tokyo, Japan), which included a DB-5MS column (30 m, 0.25 mm, 0.25 µm) (Agilent Technologies, J & W Scientific Products, Folsom, CA, USA.).Helium was used as the carrier gas and the split ratio 5:1 was employed.The initial temperature was 50 • C without hold time; the programmed rate was 10 • C for 1 min up to final temperature of 300 • C for 20 min of hold time.The injector and detector temperatures were set to 300 • C and 320 • C, respectively.The mass spectra were scanned from 29 to 800 amu.Clarification on the mass spectrum of GC-MS on each identified compound was done using the JEOL's GC-MS Mass Center System software, version 2.65a (JEOL Ltd., Tokyo, Japan)

Mycelia Growth Inhibition Test
The isolate (U61-i0-k101-z05-ta102) of P. grisea, collected from wild rice (Oryza rufipogon) grown in Southern Vietnam according to a method described in Hayashi et al. [18], was used.The fungal isolate was maintained on Potato Dextrose Agar (PDA) at 28 • C ± 2 • C. Three concentrations of 1 mg/mL, 0.5 mg/mL, and 0.1 mg/mL were prepared in sterilized distilled water.An aliquot of 1 mL of each extract or fraction was added directly into 10 mL of PDA medium and poured into 9 cm diameter sterile Petri dishes, then a 2 mm 2 of mycelium agar disc was placed in the center of the Petri dish.The dishes were then incubated at 28 • C ± 2 • C in an incubator.For the controls, the fungal inoculum was treated with distilled water.After incubating for three days, the colony diameters of the treatments and controls were measured diametrically.The inhibition was calculated according to the following equation: I = ((C − T)/C) × 100%, where I is the inhibition (%), C is the colony diameter of mycelium from the control, and T is the colony diameter of the treatments [19].The inhibition of different extracting solvents was also compared to the IC 50 value, which is the concentration (mg/mL) required to inhibit 50% mycelial growth of P. grisea.The zone of inhibition and the correspondent levels of antifungal activity on P. grisea were shown in Table 1.

Statistical Analysis
The data for mycelial growth inhibition test was analyzed by analysis of variance (ANOVA), and the mean values were compared by the Duncan's Multiple Range Test using IBM SPSS Statistics for Windows, version 20.0 (p < 0.01) (IBM cooperation, Armonk, New York).

Chemical Profiles Identified by GC-MS
In this study, GC-MS was used to analyze and identify the chemical components in different extracts, as shown in Table 2 and Figures S1-S5.There were 25 constituents identified, principally belonging to long-chain fatty acids, volatile oils, sterols, phenols, derivatives of terpenes, and amines.In details, the methanol extract included 17 compounds, whilst the number of constituents identified in hexane, ethyl acetate, 1-butanol, and water were eight, eight, two, and one, respectively (Table 2).Methanol was the most effective solvent to extract phytochemicals in bamboo leaf, followed by hexane and acetate, while 1-butanol and water showed the least efficacy.

Inhibitory Activity on Mycelia Growth of P. grisea
The antifungal activities of bamboo leaf in different extracting solvents on P. grisea were examined (Table 3; Figures S6-S10).It was found that the methanol extract was inactive on growth of P. grisea at all concentrations.At the lowest dose (0.1 mg/mL), the extracts of 1-butanol and water also showed negative results.However, the antifungal activity of ethyl acetate was weak, whilst that of hexane exhibited a moderate level of inhibition (Table 3).The aqueous extract at 0.5-1.0mg/mL revealed strong inhibition on mycelial growth of P. grisea (Table 3).The antifungal activity of different extracting solvents was also compared using the IC 50 value, which was the concentration required to inhibit 50% growth of P. grisea.Thus, the lower IC 50 value reflected stronger antifungal activity.Table 4 showed that the level of inhibition was the maximal with hexane (0.62 mg/mL), followed by water (0.70 mg/mL), and ethyl acetate (0.72 mg/mL), whilst methanol showed the least activity (9.71 mg/mL).Statistically, the inhibitory levels between ethyl acetate and water were not significantly different.Means with similar letter in a column were not significantly different (p < 0.01).IC 50 is the concentration (mg/mL) required to inhibit 50% mycelial growth of P. grisea.

Discussion
Many rice pathogens have shown resistance against a number of the fungicides currently available [20].The use of plant extracts to control the infestation of fungi on rice was potent and environmentally friendly compared with synthetic fungicides [21].In a previous work, Hubert et al. [8] reported that aqueous extracts of Coffee arabica at 10% and 25% (v/v) exhibited 81.12 and 89.40% inhibition on P. grisea, respectively.Suriani et al. [22] documented that the crude extract of Piper canium inhibited mycelial growth of P. grisea.Several phenolic acids including caffeic, chlorogenic, and ferulic acids were identified in bamboo shaving extracts and were found to correlate to antioxidant activity [12].Several C-glycosyl flavonones such as orientin, isoorientin, vitexin, and isovitexin [23], and phenolic acids including caffeic, chlorogenic, and ferulic acids [12] were detected in bamboo leaf extract and correlated to antioxidant activity.Bamboo shoot exerted antibacterial (S.aureus, B. subtilis, P. aeruginosa, and E. coli) and antifungal (Fusarium oxysporum) properties.Chemicals including tannins, steroids, phenols, glycosides, flavonoids, carbohydrates, and proteins were identified and were responsible for the antimicrobial activity [24].Amadioha [25] reported that the growth of rice blast fungus was significantly suppressed by water and ethanol extracts of the leaves and oil extracts of neem seeds.
Among the compounds identified by GC-MS (Table 2), phytol has been reported to have antimicrobial [26,27] and anti-inflammatory activities [28].Shukla and Dwivedi [29] described the inhibitory effect of phenol against growth of Fusarium oxysporum.Salehan et al. [30] noted that an ethyl acetate extract of Cosmos caudatus suppressed the spore formation of Phytophthora palmivora, which caused black pod disease of cocoa.In this study, hydrazinecarboxamide, a compound exhibiting antibacterial [31] and antimycobacterial [32] activities, was detected in the hexane extract.Recently, some novel chemicals have been synthesized such as azaloes and N-(substituted)-2-isonicotionoylhydrazinocarbothioaide which showed promising antimicrobial activities [33,34].However, many plant extracts were effective against pathogenic fungi of cucumber [35], and some other economically important crops including rice, soybean, tomato, sesame, potato, and watermelon [30,36], suggesting that natural products were more favorable.In this study, among the different extracting solvents, hexane was the most active, followed by ethyl acetate and water.1-Butanol showed negligible inhibition as compared with ethyl acetate and water, whereas methanol did not show any antifungal activity (Tables 3 and 4; Figures S6-S10).
Although the chemicals identified by GC-MS were most abundant in the methanol extract (17 compounds), the other extracting solvents included fewer constituents (eight, eight, two, and one for hexane, ethyl acetate, 1-butanol, and water, respectively) (Table 2).It was concluded that the number of chemicals identified was not proportional to the level of antifungal activity observed on P. grisea (Tables 2-4).The polarities of hexane, ethyl acetate, 1-butanol, methanol, and water were 0.009, 0.228, 0.586, 0.762, and 1.000, respectively.Both hexane and ethyl acetate showed strong inhibition on the growth of the harmful fungus P. grisea, indicating that non-polar compounds in bamboo leaf might be responsible for the antifungal activity.Based on the GC-MS results, the presence of the identified constituents was not water-soluble (Table 2).It appears that water-soluble compounds have not yet been detected by the use of GC-MS in this study.Because the ethyl acetate and water extracts showed similar antifungal activity (Table 4), it was proposed that water-soluble constituents may also be involved in the inhibition of bamboo leaf on P. grisea.Further analytical instruments such as LC-MS (liquid chromatography-mass spectrometry), and CC (column chromatography) should be applied to isolate and identify potent water-soluble chemicals in bamboo leaf.In addition, the inhibition of each chemical identified by this study, as well as their mixture, on growth of P. grisea should be investigated.
Although hexane, ethyl acetate, and butanol were effective in the search for potent phytochemicals, the aqueous extract of bamboo leaf showed strong inhibition on the growth of the harmful fungus (Table 4).This evidence revealed that bamboo leaf was a promising natural source for biological control of P. grisea.Rice farmers, especially in developing countries, may extract bamboo leaf with water to utilize as a cheap, convenient, and environmentally friendly material to reduce the yield loss caused by this rice blast disease.

Conclusions
The findings of this study highlighted that bamboo leaf appeared to be a promising source to manage the infestation of P. grisea in rice production.The analysis by GC-MS showed that non-polar constituents in bamboo leaf may be responsible for the antifungal activity, although water-soluble compounds may also play a role.Purification and isolation of potent phytochemicals should be conducted to further understand the possibility of using bamboo leaf for the biological control of P. grisea.

Table 1 .
Zone of inhibition and correspondent levels of inhibitory activity.

Table 2 .
Chemical profile in different extracting solvents identified by GC-MS.

Table 3 .
Antifungal activity of bamboo leaf extracts on mycelial growth (mm) of P. grisea.

Table 4 .
IC 50 values of different extracts on mycelial growth of P. grisea.