Eur J Plant Pathol DOI 10.1007/s10658-014-0485-7

Control efficacy against rice sheath blight of Platycladus orientalis extract and its antifungal active compounds Haihua Wang & Jinyu Wang & Xixu Peng & Pinglan Zhou & Ningning Bai & Jiao Meng & Xiaobo Deng

Accepted: 16 July 2014 # Koninklijke Nederlandse Planteziektenkundige Vereniging 2014

Abstract The control efficacy of Platycladus orientalis extract against Rhizoctonia sonali Kühn, the causal agent of rice sheath blight, was evaluated by pot experiments under greenhouse conditions, and the antifungal compounds were isolated and identified through antifungal bioassay-guided fractionation using R. sonali as a tested fungus. The results indicate that the extracts from P. orientalis exhibited a significant reduction in the severity of rice sheath blight. The petroleum ether fraction partitioned from the ethanolic crude extract, showing the highest antifungal activity, was further separated, and two diterpenoid compounds with antifungal property, totarol and sclareol, were isolated and identified from the active subfractions. Totarol and sclareol possessed antifungal activity against most of the tested fungal pathogens of cereal crops such as R. solani, R. cerealis and Fusarium graminearum, indicating a similar broad antifungal spectrum. These findings suggest that the P. orientalis extract and its derived active compounds may be promising candidate agents for controlling plant fungal diseases like rice sheath blight.

H. Wang (*) : J. Wang : X. Peng : P. Zhou : N. Bai : J. Meng : X. Deng School of Life Science, Hunan University of Science and Technology, Key Laboratory of Integrated Management of the Pests and Diseases on Horticultural Crops in Hunan Province, Xiangtan 411201 Hunan, China e-mail: [email protected]

Keywords Rice sheath blight . Rhizoctonia solani . Platycladus orientalis . Antifungal activity . Antifungal compounds

Introduction Sheath blight disease, caused by Rhizoctonia solani Kühn, is one of two major rice fungal diseases occurring in all rice cultivated areas worldwide, especially in the intensified production systems where high-yielding, short-stalked, high-tillering and fertilizer-tolerant rice cultivars are applied. Its importance only ranks after, and often rivals, rice blast, caused by Magnaporthe oryzae (Lee and Rush 1983). It is estimated that the disease causes a yield loss ranged from 2.5 to 50 % (Rabindran and Vidhyasekaran 1996). To date, no rice varieties have been screened to be immune to R. solani, although different levels of resistance have been documented in rice (Srinivasachary et al. 2011). The resistance levels of popular rice varieties or hybrid accessions applied in agricultural production in China are somewhat low. Moreover, R. solani is a highly saprophytic, soil borne fungus with sclerotia viable in soil for several years, thus increasing the difficulty of control of this disease. Application of fungicides is the major measure for controlling rice sheath blight. Jinggangmycin, in which validamycin is the key ingredient, has been extensively used as a unique fungicide in China for over three decades and also used in other Asian countries (Zhang et al. 2009). However, extensive and continuous use of a

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single chemical may lead to undesirable effects such as residual toxicity and environmental pollution, and also increases the risk of resistance development (Brent and Hollomon 1998). For example, the recommended usage amount of jinggangmycin for the management of rice sheath blight is increasing from twice per growing season and 1.5 kg/ha three decades ago to the present 3–4 times per year and 4.5–6.0 kg/ha in China. Field resistant strains of R. sonali to jinggangmycin were documented to occur sporadically in Henan and Fujian province in China (Zhang et al. 2009). Moreover, due to its possible adverse effects on residual toxicity to human and environment, the usage of validamycin has been banned in the European Union for over 10 years. Therefore, it is of urgency to develop environmentally friendly, lowl residual and effective alternative methods for the management of the disease. Great attention has been paid to screen natural products of plant origin, either directly as crude preparations, or as pure compounds for plant disease control, due to easy biodegradability, low toxicity and minimum residues in the agro-ecosystem (Kagale et al. 2004; Aye and Matsumoto 2011). Also, the isolation and chemical modification of natural active compounds from plants is becoming one of the important strategies for development of new agrochemicals. Plants synthesize a great diversity of secondary metabolites including alkaloids, terpenoids, flavonoids, glycosides, quinones, coumarins, phenolics, essential oils and phytoalexins, some of which are known for antimicrobial activity (Fawcett and Spencer 1970; Zhou 2005). Al-Mughrabi (2003) reported that extracts from Euphorbia macroclada were found to be effective against spore germination and mycelial growth of R. solani, Verticillium dahliae, Fusarium oxysporum, Rhizopus stolonifer, and Penicillium italicum. Clerodane diterpenoids isolated from Scutellaria spp. possess inhibitory activity against F. oxysporum and V. tricorpus (Cole et al. 1991). Similarly, crude extracts and alkaloid matrine from Sophora flavescens, a Chinese traditional herb medicine, inhibit a variety of plant fungal pathogens such as Pythium aphanidermatum, P. graminicola, Gibberella zeae, Glom erella cingulata, Botrytis cinerea and F. oxysporum. Several fungicides using matrine as a main ingredient have been developed for the management of plant diseases for decades in China (Li et al. 2006). There is evidence from earlier researchers that several plant species possess antifungal properties against the

fungus causing rice sheath blight (Kurucheve et al. 1997; Kandhari and Devakumar 2003; Pal et al. 2011). Aye and Matsumoto (2011) evaluated the inhibitory efficacy of 16 naturally available phytoextracts against R. solani in vitro, and found that leaf extracts of clove, neem, rosemary and pelargonium were potential plant products to control rice sheath blight. Two essential oils from Lippia geminata and Cymbopogon jwarancusa can effectively reduce the growth and sporulation of pathogens of rice sheath blight and brown spot disease in vitro, and appear to be good candidates for the control of these pathogens in appropriate formulations (Deka Bhuyan et al. 2010). However, few attempts have been made for the management of rice sheath blight by bioactive plant extracts and metabolites under greenhouse and field conditions other than applying with fungicides (Kagale et al. 2004). Moreover, few antifungal compounds of plant origin against the rice sheath blight fungus have been isolated and identified so far. Previously, leaf or root extracts of 15 plant species, belonging to various families, were evaluated for their antimicrobial activity against R. sonali, the causal agent of rice sheath blight. Among them, Platycladus orientalis was found to be highly effective in inhibiting the mycelial growth of the fungus in vitro, and its antifungal activity was effectively enriched in the ethanolic extract. For isolation and identification of antifungal compounds, the ethanolic crude extract was further successively fractioned by solvents with different polarities such as EtOAc, n-butanol, petroleum ether and water, and their antifungal activities against R. solani were evaluated. The petroleum ether fraction exhibited the highest reduction in the radial mycelial growth of the pathogen, while the lowest inhibition of growth reduction was recorded by the aqueous fraction (Wang et al., 2011). P. orientalis, a Cupressaceae plant native to China, Korea, and the Russian Far East, is used most often in formal hedges, screens and windbreaks, or as specimen plantings.Little information is available on its antimicrobial property of extracts and constitutes. The present study is aimed to: (1) evaluate the potential of P. orientalis extracts for controlling rice sheath blight under greenhouse conditions, (2) isolate and identify antifungal compounds from the active fractions of P. orientalis, and (3) to test the in vitro antifungal activity of the isolated compounds on several important fungal pathogens of cereal crops such as R. solani, M. oryzae, R. cerealis, Gaeumannomyces graminis var. graminis and F. graminearum. Additionally, the

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structural changes in fungal mycelia caused by the antifungal compounds were also observed by light microscopy.

Materials and methods Plant material Branches and leaves of P. orientalis cv. sieboldii were collected from the Botanical Garden of the University campus during April to June, 2009. The plant specimen was identified by Associate Professor Liu Bing-Rong at Department of Life Science, Hunan University of Science and Technology. The selected plant samples were washed, dried at 50 °C to constant weight and ground to fine powders (60 mesh). Fungal pathogens R. solani (provided by Prof. Zhou Er-xun of the Department of Plant Pathology, South China University of Science and Industry), M. oryzae (provided by Dr. Hao Zhong-na at Institute of Plant Protection and Microbiology, Zhejiang Academy of Agricultural Sciences), R. cerealis, G. graminis var. graminis and F. graminearum were grown on Potato Dextrose Agar (PDA) medium for 2–5 days at 25–28 °C. Preparation of plant extracts 3 kg of weighted powder was soaked in 95 % ethanol 1:10 (w/v) in a hermetically closed glass vessel under occasional shaking at room temperature for 48–72 h each time. The extraction was repeated three times. The three extracts were combined, filtered and evaporated on a rotary evaporator under reduced pressure at 60 °C. The resulting dried extract was suspended in warm water (1:1, w/v), and partitioned with petroleum ether (1:1, w/v) twice to yield 47.8 g fraction.

evaporator. Fr. 8, showing the most effective antifungal activity, was further separated into eight sub-fractions (Fr. 8–1–8) by thin layer chromatography (TLC, Merck, Germany). To obtain pure compounds, preparative TLC (PTLC) plate 20×20 (Sigma-Aldrich, China) was applied to isolate active compounds from the active subfractions based on antifungal activity evaluation. The eluted solvents were petroleum ether, EtOAc, and acetone in different proportions. The separation scheme of the antifungal compounds from the petroleum ether fraction is shown in Fig. 1. Five compounds (Com. 1, 56 mg; Com. 2, 43 mg; Com. 3, 27 mg; Com. 4, 19 mg; Com. 5, 62 mg) were obtained. The purities of the isolated active compounds were analyzed by gas chromatography (GC) using the Shimadzu 2014 GC equipped with a Shimadzu Hicap series CBP1 column (30 m×0.25 mm×0.25 μm) and a flame ionized detector (FID). Helium gas was used as a carrier gas with a flow rate of 1 ml/min. The column temperature was set at 80 °C maintained for 5 min, and then programmed ramp to 260 °C, maintained for 10 min, with a total time of 33 min. The temperature of injector and detector was set at 250 and 260 °C, respectively, and the injection volume was 1 μl. The compound purities were estimated by area normalization method. The structures of the active compounds were identified by different spectroscopic techniques (EI-MS, NMR, UV–VIS and FT-IR). EI-MS was conducted with a Finnigan LCQ Advantage MAX mass spectrometer. Full scan EI-MS were acquired over a range of m/z 20– 500 with ESI (+) ionization mode, 70 eV transmission voltage and 45 psi nebulizer pressure. All NMR spectra were recorded on a Bruker AV-II 500 MHz NMR

Isolation and identification of antifungal compounds The petroleum ether fraction was subjected to silica gel column (200–300 mesh, Qingdao Ocean Chemical Plant, China) and eluted with gradient hexane/ ethyl acetate (EtOAc) from 1:0 (v/v) to 3:2 (v/v) to yield eight fractions (Fr. 1–8). Excessive solvents in each partitioned fraction were removed by a rotary

Fig. 1 Separation scheme of compounds from the petroleum ether fraction of P. orientalis

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spectrometer. TMS was used as an internal reference for 1 H and 13C chemical shifts, and CDCl3 was used as solvent. The UV–vis of the sample (0.1 mg/ml) solution was prepared in methanol at the range of 200–600 nm. FT-IR spectra were recorded on a Perkin-Elmer spectrometer. Mixed with KBr and pressed into pellets, the samples were analyzed by infrared spectrometer scanning from 4,000 to 450/cm. Antifungal assay in vivo Antifungal activity of plant extracts was evaluated by agar dilution method (Quiroga et al. 2001). Briefly, 2 day-old fungal culture plugs (0.5 cm in diameter) were obtained and placed at the centre of Petri dishes (9 cm) in PDA culture media containing extracts (dissolved in acetone) at a final concentration of 0.5 mg/ml. PDA plates containing acetone instead of extracts was used as control. The cultures were incubated at 25 °C under dark. The radial growth of mycelia was measured after 36–48 h of inoculation when the mycelia growth on control plates reached the inner edge. The percentage of inhibition was calculated using the following formula: Relative efficacy of inhibition (%) = (mycelial growth of the control-mycelial growth of the treatment)/mycelial growth of the control × 100. Antifungal activity of the isolated compounds was evaluated by paper disk-agar diffusion method as described by Matook and Toshihiko (2006) with modifications. Briefly, 2-day-old mycelial plugs (0.5 cm diameter) of R. solani were placed at the center of Petri dishes (9 cm diameter) containing 15 ml PDA medium. Filter paper discs (Whatman No. I, 0.6 cm diameter) impregnated with 0.1 and 0.5 mg/disc compounds were equidistantly placed on the PDA plates (2.5 cm away from the mycelial plug). Carbendazim, a commercial fungicide, was used as positive control. Discs soaked in pure solvent of acetone were used as negative control. The inhibition zones around the discs were recorded after 36 h of inoculation. Antifungal effects of isolated compounds on fungal mycelium morphology After the inhibition zones of fungal mycelia were measured, a small section of mycelia was prepared from the edge of control mycelia or mycelia treated with compounds, and examined with bright-field optics using a light microscope (XS-402, Ningbo, China). Images of

mycelial deformities were captured using a digital camera. Control efficacy under greenhouse conditions A pot experiment was carried out to test the control efficacy of P. orientalis extracts against rice sheath blight. Seeds of rice (Oryza sativa, Zhonghua 11) were sterilized with 0.1 % HgCl2 for 10 min and washed extensively with distilled water. After 24 h of imbibition in distilled water, the seeds were sown in trays of soil (pH 5.2; Organic matter 21.6 g/kg). The soil was mixed with composite fertilizer at a mass ratio of 0.15 N: 0.10 P2O5: K2O 0.15 as a base fertilizer. When the second leaf was fully expanded (about 15 days after sowing), five uniform seedlings were transplanted to each ceramic pot (diameter 30 cm, 40 cm height) filled with 4 kg soil. Culture conditions were set as follows: 26–28 °C (day)/20–22 °C (night), natural illumination, and relative humidity between 80 and 85 %. One month after rice transplanting, each plot was evenly spread with 5 g wet grain inocula of R. sonali for inoculation. The grain inocula were prepared as previously (Xie et al. 2012). Two days before or after inoculation with the pathogen, the plants were sprayed with 1.0 mg/ml P. orientalis extracts (containing 0.01 % SDS as a surfactant), respectively. Plants sprayed with 0.01 % SDS and 0.01 % jinggangmycin (plus 0.01 % SDS) were served as negative and positive controls, respectively. Three replications were set for each treatment, and each replication consisted of five pots with completely a randomized design. The disease severity was recorded when withered or dead rice plants began to occur in the negative control, and assessed using the score chart 0–9 scale (IRRI 1996). Disease severity and control efficacy were calculated according to Yang et al. (2009): Disease severity (%) = [∑(The number of diseased plants in a certain index × Disease index)/(Total number of plants investigated × The highest disease index)]/100. Control efficacy (%) = [(Disease severity of controlDisease severity of treated group)/Disease severity of control] × 100. Statistical analysis All results presented are the mean of three independent replicates. The data were analyzed by analysis of variance (ANOVA) using SPSS for Windows version 13.0

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(SPSS Inc, Chicago, USA). The mean values were compared by Duncan’s multiple range test at P<0.05 level.

Results Control efficacy of P. orientalis extracts against rice sheath blight in greenhouse To ascertain whether the ethanolic crude extract and the petroleum ether fraction could protect rice plants against sheath blight, we applied a pot experiment to explore its control efficacy under greenhouse conditions. Data in Table 1 shows that either pre- or post-inoculation spray with 1.0 mg/ml ethanolic crude extract or petroleum ether fraction exhibited a significant reduction in sheath blight compared with the non-treated control, but lower than that of commercial fungicide Jinggangmycin (0.01 %). There was no statistically significant difference in the control efficacy between pre- and postinoculation sprays with the same extract or fraction, although slightly higher control efficacy was observed in the pre-inoculation application. When pre-inoculation sprayed with the ethanolic crude extract, the disease severity of the plants was reduced by 57.9 %, and the control efficacy reached 65.4 %. The petroleum ether fraction showed significantly higher control efficacy compared with that of the ethanolic crude extract.

Antifungal activity in vitro of petroleum ether fractions To isolate and identify antifungal compounds from P. orientalis, the petroleum ether fraction that exhibited the highest antifungal activity in vitro (Wang et al., 2011) and control efficacy against rice sheath blight fungus (Table 1) was further fractioned into eight fractions (Fr. 1–8) by silica gel column chromatography. All fractions were tested for antifungal activity against R. solani in vitro. As shown in Fig. 2, the fractions 2– 8 (Fr. 2–8) reduced the mycelial development of R. solani with varying degrees at 0.5 mg/ml concentration. Among the fractions tested Fr. 8 exhibited the

Table 1 Control efficacy of P. orientalis extracts at 1.0 mg/ml concentration against rice sheath blight under greenhouse conditions Plant extracts

Pre-inoculation spray

Post-inoculation spray

Disease Control Disease Control severity efficacy severity efficacy (%) (%) (%) (%) Ethanolic crude extract

30.6b

65.4c

35.0b

62.7c

c

b

c

75.9b

d

Petroleum ether fraction 19.4 0.01 % Jinggangmycin (positive control) 0.1 % SDS (negative control)

d

78.1

a

22.6

16.3

82.7

20.1

80.3a

88.5a

0d

93.6a

0d

Values within the same column followed by different letters were significantly different based on Duncan’s multiple range test (P<0.05)

Fig. 2 Inhibitory effects of the petroleum ether fraction of P. orientalis on the mycelial growth of R. solani. The radial growth of mycelia was measured after 36–48 h of inoculation. a Photos; b Relative inhibition (%). Bars with different letters are significantly different (P<0.05) according to Duncan’s multiple range test. Fr, fraction

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highest mycelial growth inhibition (76.6 %), followed by Fr. 4 (48.7 % inhibition). However, Fr. 1 (mostly nature pigment substances with low polarity) had a slightly promoting effect on the mycelial growth of R. solani. Subsequently, Fr. 8 was further fractioned into eight sub-fractions (Fr. 8–1–8). All sub-fractions exhibited antifungal activity to varying degrees (Fig. 3). Fr. 8–2 showed the highest reduction (79.6 %) in the mycelial growth of R. solani followed by Fr. 8–1 and Fr. 8–8 (76.8 % and 73.9 %, respectively). The lowest inhibition (38.1 %) was recorded by Fr. 8–6. Thus, Fr. 8–1, 8–2 and 8–8 were selected for isolation and identification active compounds in further experiments.

Fig. 3 Inhibitory effects of the subfractions from the petroleum ether fraction of P. orientalis on the mycelial growth of R. solani. The radial growth of mycelia was measured after 36–48 h of inoculation. a Photos; b Relative inhibition (%). Bars with different letters are significantly different (P< 0.05) according to Duncan’s multiple range test. Fr, fraction

Compounds with antifungal activity Five compounds were isolated by PTLC from the subfractions 8–1, 8–2 and 8–8. Com. 2 and Com. 4 were chosen for further structure elucidation as they exhibited mycelial growth inhibition against R. solani with obvious inhibition zones at 0.1 and 0.5 mg/disc concentrations (Fig. 4). In the control plates (without active compounds), the mycelia of R. solani appeared uniform, whereas in the R. solani plates treated with the active compounds, the most of R. solani mycelia became swollen or collapsed, twisted. The mycelial malformation caused by Com. 2 (as identified below as totarol) was more severe than that by Com. 4 (as identified below as sclareol) (Fig. 5). Similar results were observed with P. orientalis extracts (Wang et al. 2011). However, no antifungal activity against the fungus was achieved by Com. 1, Com.3, and Com. 5 (data not shown). The retention time of Com. 2 and Com. 4 on gas chromatography was 16.039 and 15.568 min, respectively, at the chromatographic conditions described above. Their purity reached to 97.6 and 96.6 %, respectively (data not shown), indicating that they could be used for subsequent structural identification by spectroscopic methods. The physical and chemical properties of the two isolated compounds are as follows: Com. 2 was isolated as a light yellow powder. Its molecular formula was assigned as C20H30O by EI-MS measurements and NMR spectroscopic analyses. Molecular weight 286.23; UV–vis (CH3OH) lmax (nm): 222, and 279 (benzene ring); IR (KBr) (v/cm): 3585.38, and 3460.49 (hydroxyl group), 2921.60 (methylene group), 1372.67 (methyl group), 1483.66, 1470.56, and 1455.17 (benzene ring), 1266.65, and 1177.00 (C-O bond of phenol). EI-MS m/z: 286.2 [M + H]+,285.2 [M] +,271.1 [M-CH3]+. 1H NMR (CDCl3, 500 MHz), %: 0.912 (3H, s, H-19), 0.944 (3H, s, H-18), 1.174 (3H, s, H-20), 1.329–1.356 (6H, t, J = 6.5, H-15, 16), 1.448–1.473 (1H, d, J = 12.5, H-5), 1.887–1.930 (1H, t, J = 9, H-6’), 2.212–2.237 (1H, d, J = 12.5, H-1’), 2.712–2.784 (1H, m, H-7), 2.914–2.961 (1H, dd, J = 6.5, H-7’), 3.274–3.301 (1H, br, H-15), 4.436 (1H, s, OH), 6.502–6.519 (1H, d, J = 8.5, H-12), 6.986–7.003 (1H, d, J = 8.5, H-11). 13C NMR (CDCl3,125 MHz), %c: 19.32(C-6), 19.46 (C-2), 20.31 (C-16), 20.33 (C-17), 21.55 (C-19), 25.15 (C-20), 27.11 (C-17), 28.73 (C-7), 33.21 (C-4), 33.23 (C-18), 37.66 (C-10), 39.56 (C-1), 41.54 (C-3), 49.53 (C-5), 114.26 (C-12), 122.96 (C-11),

Eur J Plant Pathol Fig. 4 Inhibitive effects of Compounds 2 (a) and 4 (b) on the mycelial growth of R. solani as evaluated by paper disk-agar diffusion method: a, 0.1 mg/disc; b, 0.5 mg/disc; CK, control

130.95 (C-14), 133.96 (C-8), 143.17 (C-9), and 151.91 (C-13). By spectroscopic data and comparing the MS, 1 H- and 13C-NMR information with known compounds, Com. 2 was identified as totarol (Fig. 6a), 14Isopropylpodocarpa-8, 11, 13-trien-13-ol (Ying and Kubo 1991). Com. 4 was isolated as a white powder. Its molecular formula was assigned as C20H30O by EI-MS measurements and NMR spectroscopic analyses. UV–vis (CH 3 OH) lmax (nm): no signals; IR (KBr) cm): 3364.79 (hydroxyl group), 1643.80 (C = C), 2924.98, and 2937.91 (methylene group), 1457.07 (methyl group), 1119.52 (C-O bond of alcohol), and 1083.09 (-OH of alcohol). EI-MS m/z: 291.3 [M-H2O + H]+, 290.3 [M-H2O]+, 275.1 [M-H2O-CH3]+; 1H NMR (CDCl 3 , 500 MHz), %: 0.783 (6H, s, H-18, 19), 0.861(3H, s, H-20), 0.905–0.910(2H, m, H-11), 1.156 (3H, s, H-17), 1.276 (3H, s, H-16), 1.601–1.668 (6H, m, H-1, 2, 3, 6, 12, 12’), 1.829–1.854 (1H, m, H-7’), 5.018–5.042 (1H, m, H-15), 5.197–5.234 (1H, m, H-15’), and 5.905–5.961 (1H, t, J=10, H-14). 13C NMR (CDCl3,125 MHz), %c: 15.36 (C-11), 18.39 (C-6), 18.98 (C-2), 20.45 (C-20), 21.45 (C-18, 19), 24.14 (C-17), 26.82 (C-16), 33.19 (C-10), 33.35 (C-4), 39.21 (C-1), 39.63 (C-7), 41.96 (C-12), 44.22 (C-3),

56.03 (C-5), 61.64 (C-9), 73.49 (C-13), 74.73 (C-8), 111.00 (C-15), and 146.22 (C-14). On the basis of spectral examination and comparison with literature data (Bailey et al. 1974; Bai 2004), Com. 4 was identified as sclareol (Fig. 6b), (1R, 2R, 8aS)-Decahydro1-(3-hydroxy-3-methyl-4-pentenyl)-2, 5, 5, 8atetramethyl-2-naphthol. Antifungal activity of totarol and sclareol against fungal pathogens The antifungal activity of the two isolated compounds at 0.1 mg/disc concentration was evaluated against two rice fungal pathogens, R. solani and M. oryzae, and three wheat fungal pathogens, R. cerealis (sheath blight), Gaeumannomyces graminis var. graminis (take-all disease) and Fusarium graminearum (scab). As shown in Table 2, totarol possessed strong antifungal activity against R. solani and R. cerealis (inhibition zone 13.7, 12.4 mm, respectively), moderate antifungal activity against F. graminearum (inhibition zone 6.6 mm), and weak antifungal activity against G. graminis var. graminis (inhibition zone 2.1 mm). However, no mycelial growth inhibition was observed against M. oryzae. Similarly, sclareol exhibited different reductions in

Fig. 5 Mycelial morphology of R. solani on agar without compounds (a), with totarol (b), and sclareol (c) at 0.1 mg/disc concentration

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Fig. 6 Structures of isolated compounds from antifungal fractions of P. orientalis. a, totarol; b, sclareol

mycelial growth of the tested pathogens. The observed values of inhibition zones can be ranked in the following descending order: F. graminearum > R. solani > R. cerealis > M. oryzae. However, it failed to show any inhibition effects against G. graminis var. graminis. These results indicate that both totarol and sclareol have a broad spectrum of antifungal activity, and show strong antifungal activity against R. solani, R. cerealis and F. graminearum, which are soil borne fungal pathogens of cereal crops.

Discussion Plant-derived substances have potential to control some of soil borne phytopathogens, and may represent a promising strategy for controlling such plant diseases (Chutia et al. 2009; Deka Bhuyan et al. 2010). Rice sheath blight, caused by the soil borne fungus, R. solani, is emerging as a very destructive disease under favourable weather conditions in rice-cultivation areas of the world, which ultimately results in substantial yield losses. Alternative natural active materials to the commonly used fungicides are receiving increased

attention to control the disease in a more environmentally friendly way. Accumulating evidence indicates that extracts from several plants, such as Azadirachta indica, Allium sativum, L. geminata, C. jwarancusa, Syzygium aromaticum, Rosmarinus officinalis, Pelargonium spp., possess antifungal or fungistatic activity against the rice sheath blight fungus, and appear to be potential alternatives for the control of this disease (Kandhari and Devakumar 2003; Deka Bhuyan et al. 2010; Aye and Matsumoto 2011). However, the actual control efficacy of plant extracts against rice sheath blight has not been extensively investigated under greenhouse and field conditions, and very few materials are commercially available. In the present study, we evaluated the control effect of P. orientalis extracts against rice sheath blight by pot experiments in a greenhouse. The results indicate both the ethanolic crude extract and the petroleum ether fraction of P. orientalis can significantly reduce the disease severity (Table 1), suggesting a promising potential for the management of rice sheath blight. Despite of higher effectiveness achieved by the commercial fungicide jinggangmycin (0.01 %), P. orientalis extract as a potential alternative for disease control is still attractive when its properties of easy availability, low cost, and less environmental risk are taken into consideration. Mechanisms of disease suppression by plant products may either act on pathogens directly, or induce systemic resistance in host plants resulting in reduction of disease development (Amadioha 2000; Kagale et al. 2004; Latha et al. 2009). Treatment with P. orientalis extracts results in turgidity or shrinkage, twist and even collapse of R. solani mycelia (Wang et al. 2011), suggesting that they may contain active constitutes which are toxic to the pathogen. In the present study, two compounds with antifungal activity, totarol and sclareol,

Table 2 Growth inhibition of totarol and sclareol against plant fungal pathogens using agar disc diffusion methods at 0.1 mg/disc concentration Fungi tested

Rhizoctonia solani

Inhibition zone (mm) Totarol

Sclareol

Carbendazim (positive control)

Acetone (negative control)

13.7a

10.3a

14.7c

–

Magnaporthe oryzae

–

3.2

16.3b

–

Rhizoctonia cerealis

12.4a

5.7b

11.0d

–

–

19.6ab

–

22.5a

–

Gueumannomyces graminis

2.1c

Fusarium graminearum

6.6b

c

11.5a

c

-, no inhibition observed. Values within the same column followed by different letters were significantly different based on Duncan’s multiple range test (P<0.05)

Eur J Plant Pathol

were isolated (Fig. 6; Table 2), confirming this possibility. However, the antifungal effect exerted by P. orientalis extracts depends on the synergism of many compounds, and a single component from P. orientalis has limited functions in the total antifungal activity. Therefore, totarol and sclareol are only partially responsible for the in vitro antifungal property and the control efficacy of P. orientalis extracts against R. solani as evaluated by the pot culture under greenhouse conditions. Interestingly, totarol and sclareol exhibited the growth inhibition of three soil borne fungal pathogens of crops, i.e., R. solani, R. cerealis and F. graminearum (Table 2), suggesting their application potentials for controlling certain soil borne diseases of crops. Totarol and sclareol are categorized as diterpenoids, important resources of plant-derived antimicrobial substances (Zhou 2005). Previous investigations demonstrated that the resistance of some trees against microorganisms and insects is associated with high concentrations of phenolic terpene compounds (Becerra et al. 2002; Solís et al. 2004). The antifungal activity of diterpenoids may be attributed to the phenolic group joined with a single hydroxyl group, which confers lipophilicities and acidity (Laks and Pruner 1989). Our results are in line with these findings. Totarol was originally isolated from Podocarpus totara, and has also been identified from several species of Cupressaceae such as Thujopsis dolabrata and Pilgerodendron uviferum (Solís et al. 2004; Yamaji et al. 2007). In our study, it is also detected in leaves and branches of P. orientalis, which belongs to the same family with T. dolabrata and P. uviferum. Totarol possesses unique antimicrobial and therapeutic properties. Yamaji et al. (2007) observed that totarol, as a main antifungal compound of T. dolabrata seeds, plays an important role in selecting fungi on root surface of seedlings in the early growth stage. In line with this observation, the present results show that totarol exhibited in vitro antifungal activity against R. solani, R. cerealis and F. graminearum (Table 2). Sclareol, first isolated from clary sage (Salvia sclarea), is a labdane-type of diterpene produced by a few of plants including Nicotiana species. Evidence reveals that it possesses antimicrobial activity against several plant pathogens (Bailey et al. 1974; Bailey et al. 1975; Kennedy et al. 1992; Jackson and Danehower 1996). Bai (2004) found that sclareol and its derivatives exhibited significant inhibitory effects on the mycelial

growth of Gibbere zeae, Blumeria graminis, F. solani and Alternaria longipes. Sclareol also exhibits the potential for activation of induces expression of defenserelated genes, thus conferring induced resistance against pathogens in plants (Campbell et al. 2003; Seo et al. 2012). In our study, sclareol is isolated from P. orientalis, and exerts inhibitory effects on the mycelial growth of certain fungi including R. solani. This is the first report on the isolation of antifungal compound, sclareol, from a wood tree (P. orientalis). Taken together, our findings clearly demonstrate that P. orientalis extracts exhibit significant control efficacy against rice sheath blight. The isolated two compounds, totarol and sclareol have in vitro antifungal activity. This suggests that P. orientalis extract and its derived natural compounds have a great potential to be developed as environmentally benign antifungal agents for controlling plant fungal diseases like rice sheath blight. However, further experiments should be performed to ascertain the efficacy in field conditions for facilitating practical use. Moreover, these compounds are required further investigations to elucidate their antifungal mechanisms before pharmaceutical application. Acknowledgments We thank Dr. Yu Xian-Yong from Hunan University of Science and Technology for NMR analysis. This research was funded by National Natural Science Foundation of China (Grant No. 31171803, 31301617) and Hunan Provincial Education Department (12C0129).

References Al-Mughrabi, K. I. (2003). Antimicrobial activity of extracts from leaves, stems and flowers of Euphorbia macroclada against plant pathogenic fungi. Phytopathologia Mediterranea, 42, 245–250. Amadioha, A. C. (2000). Controlling rice blast in vitro and in vivo with extracts of Azadirachta indica. Crop Protection, 19, 287–290. Aye, S. S., & Matsumoto, M. (2011). Effect of some plant extracts on Rhizoctonia spp. and Sclerotium hydrophilum. Journal of Medicinal Plants Research, 5(16), 3751–3757. Bai, H. J. (2004). Studies on synthesis and bioactivity of sclareol derivatives (in Chinese). Yangling, China: Northwest SciTech University of Agriculture and Forestry, thesis for Master degree. Bailey, J. A., Vincent, G. G., & Burden, R. S. (1974). Diterpenes from Nicotiana glutinosa and their effect on fungal Growth. Journal of General Microbiology, 85, 57–64. Bailey, J. A., Carter, G. A., Burden, R. S., & Wain, R. L. (1975). Control of rust disease by diterpenes from Nicotiana glutinosa. Nature, 255, 328–329.

Eur J Plant Pathol Becerra, J., Flores, C., Mena, J., Aqueveque, P., Alarcón, J., Bittner, M., Hernández, V., Hoeneisen, M., Ruiz, E., & Silva, M. (2002). Antifungal and antibacterial activity of diterpenes isolated from wood extractables of Chilean Podocarpaceae. Boletin De La Sociedad Chilena De Quimica, 47, 151–157. Brent, K. J., & Hollomon, D. W. (1998). Fungicide resistance: the assessment of risk. In FRAC Monograph 2 (pp. 1–48). Brussels, Belgium: Global Crop Protection Federation (Now CropLife International). Campbell, E. J., Schenk, P. M., Kazan, K., Penninckx, I. A. M. A., Anderson, J. P., Maclean, D. J., Cammue, B. P. A., Ebert, P. R., & Manners, J. M. (2003). Pathogen-responsive expression of a putative ATP-binding cassette transporter gene conferring resistance to the diterpenoid sclareol is regulated by multiple defense signaling pathways in Arabidopsis. Plant Physiology, 133, 1–13. Chutia, M., Deka Bhuyan, P., Pathak, M. G., Sarma, T. C., & Boruah, P. (2009). Antifungal activity and chemical composition of Citrus reticulata Blanco essential oil against phytopathogens from North East India. LWT–Food Science and Technology, 42, 777–780. Cole, M. D., Bridge, P. D., Dellar, J. E., Fellows, L. E., Cornish, M. C., & Anderson, J. C. (1991). Antifungal activity of neoclerodane diterpenoids from Scutellaria. Phytochemistry, 30(4), 1125–1127. Deka Bhuyan, P., Chutia, M., Pathak, M. G., & Baruah, P. (2010). Effect of essential oils from Lippia geminata and Cymbopogon jwarancusa on in vitro growth and sporulation of two rice pathogens. Journal of the American Oil Chemists’ Society, 87, 1333–1340. Fawcett, C. H., & Spencer, D. M. (1970). Plant chemotherapy with natural product. Annual Review of Phytopathology, 8, 403– 419. IRRI. (1996). Standard evaluation system for rice (4th ed., p. 25). Manila: International Rice Research Institute. Jackson, D. M., & Danehower, D. A. (1996). Integrated dace study: Nicotiana leaf-surface components and their effects on insect pests and diseases. In G. Kerstines (Ed.), Plant Cuticles: An Integrated Functional Approach (pp. 231– 254). Oxford: BIOS Scientific Publishers. Kagale, S., Marimuthu, T., Thayumanavan, B., Nandakumar, R., & Samiyappan, R. (2004). Antimicrobial activity and induction of systemic resistance in rice by leaf extract of Datura metel against Rhizoctonia solani and Xanthomonas oryzae pv. oryzae. Physiological and Molecular Plant Pathology, 65, 91–100. Kandhari, J., & Devakumar, C. (2003). Effect of neem oil and its fraction against sheath blight (Rhizoctonia solani Kühn) of rice. Journal of Mycopathology Research, 41, 185–187. Kennedy, B. S., Nielsen, M. T., Severson, R. T., Sisson, V. A., Stephenson, M. K., & Jackson, D. M. (1992). Leaf surface chemicals from Nicotiana affecting germination of Peronospora tabacina (Adam) sporangia. Journal of Chemical Ecology, 18, 1467–1478. Kurucheve, V., Gerard Ezhilan, J., & Jayaraj, J. (1997). Screening of higher plants for fungitoxicity against Rhizoctonia solani in vitro. Indian Phytopathology, 50, 235–241. Laks, P. E., & Pruner, M. (1989). Flavonoid biocides: Structure/ activity relations of flavonoid phytoalexin analogues. Phytochemistry, 28(1), 87–91.

Latha, P., Anand, T., Ragupathi, N., Prakasam, V., & Samiyappan, R. (2009). Antimicrobial activity of plant extracts and induction of systemic resistance in tomato plants by mixtures of PGPR strains and Zimmu leaf extract against Alternaria solani. Biological Control, 50, 85–93. Lee, F. N., & Rush, M. C. (1983). Rice sheath blight: a major rice disease. Plant Disease, 67(7), 829–832. Li, L. N., Ji, M. S., & Su, Z. Y. (2006). Research advances in use of the agricultural fungicide Sophora flavescens (in Chinese). Agrochemicals, 45(9), 581–583. Matook, S. M., & Toshihiko, S. (2006). Antioxidant and antimicrobial activities of the methanol extracts from pummelo (Citrus grandis Osbeck) fruit albedo tissues. European Food Research Technology, 224, 39–47. Pal, T. K., Bhattacharya, S., & Chakraborty, K. (2011). Induction of systemic resistance in rice by leaf extract of Cymbopogan citrus and Ocimum sanctum against sheath blight disease. Archives of Applied Science Research, 3(1), 392–400. Quiroga, E. N., Sampietro, A. R., & Vattuone, M. A. (2001). Screening antifungal activities of selected medicinal plants. Journal of Ethnopharmacology, 74, 89–96. Rabindran, R., & Vidhyasekaran, P. (1996). Development of a formulation of Pseudomonas iluorescens PfALR2 for management of rice sheath blight. Crop Protection, 15, 715–721. Seo, S., Gomi, K., Kaku, H., Abe, H., Seto, H., Nakatsu, S., Neya, M., Kobayashi, M., Nakaho, K., Ichinose, Y., Mitsuhara, I., & Ohashi, Y. (2012). Identification of natural diterpenes that inhibit bacterial wilt disease in tobacco, tomato and Arabidopsis. Plant and Cell Physiology, 53(8), 1432–1444. Solís, C., Becerra, J., Flores, C., Robledo, J., & Silva, M. (2004). Antibacterial and antifungal terpenes from Pilgerodendron uviferum (D. Don) Florin. Journal of the Chilean Chemical Society, 49, 157–161. Srinivasachary, Willocquet, L., & Savary, S. (2011). Resistance to rice sheath blight (Rhizoctonia solani Kühn) [(teleomorph: Thanatephorus cucumeris (A.B.. Frank) Donk.] disease: current status and perspectives. Euphytica, 178(1), 1–22. Wang, J. Y., Peng, X. X., Yin, B., Xie, Z. H., Gao, J., & Wang, H. H. (2011). Inhibitory effects of extracts from Platycladus orientalis cv. sieboldii on rice sheath blight fungus (in Chinese). Journal of Hunan University of Science & Technology (Natural Science Edition), 26(1), 115–117. Xie, Z. H., Gao, J., Wang, J. Y., Peng, X. X., Tang, X. K., & Wang, H. H. (2012). Isolation and identification of an antagonistic bacterium against Rhizoctonia solani, the causing agent of rice sheath blight (in Chinese). Microbiology China, 39(4), 477–485. Yamaji, K., Mori, S., Akiyama, M., Kato, A., & Nakashima, T. (2007). The antifungal compound totarol of Thujopsis dolabrata var. hondai seeds selects for fungi on seedling root surfaces. Journal of Chemical Ecology, 33, 2254– 2265. Yang, D. J., Wang, B., Wang, J. X., Chen, Y., & Zhou, M. G. (2009). Activity and efficacy of Bacillus subtilis strain NJ-18 against rice sheath blight and Sclerotinia stem rot of rape. Biological Control, 51, 61–65.

Eur J Plant Pathol Ying, B. P., & Kubo, I. (1991). Complete 1H and 13C NMR assignments of totarol and its derivatives. Phytochemistry, 30(6), 1951–1955. Zhang, C. Q., Liu, Y. H., Ma, X. Y., Feng, Z., & Ma, Z. H. (2009). Characterization of sensitivity of Rhizoctonia solani, causing

rice sheath blight, to mepronil and boscalid. Crop Protection, 28, 28381–28386. Zhou, L. G. (2005). Antimicrobial compounds in plants (in Chinese). Beijing: China Agricultural Science and Technology Press.