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Crop Protection 43 (2013) 65e71

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Crop Protection

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Anti-Rhizoctonia solani activity by Desmos chinensis extracts and its mechanismof action

Patimaporn Plodpai a, Samerchai Chuenchitt b, Vasun Petcharat b, Suda Chakthong c,Supayang Piyawan Voravuthikunchai a,*aDepartment of Microbiology and Natural Products Research Center, Faculty of Science, Prince of Songkla University, Songkhla 90112, ThailandbDepartment of Pest Management, Faculty of Natural Resources, Prince of Songkla University, Songkhla 90112, ThailandcDepartment of Chemistry and Natural Products Research Center, Faculty of Science, Prince of Songkla University, Songkhla 90112, Thailand

a r t i c l e i n f o

Article history:Received 2 March 2012Received in revised form31 August 2012Accepted 10 September 2012

Keywords:Rhizoctonia solaniDesmos chinensisElectron microscopeBioautographyMechanisms of action

* Corresponding author. Tel./fax: þ66 7444 6661.E-mail address: supayang.v@psu.ac.th (S.P. Voravu

0261-2194/$ e see front matter Crown Copyright � 2http://dx.doi.org/10.1016/j.cropro.2012.09.004

a b s t r a c t

The objectives of this study were to investigate the mechanisms of action and to evaluate the potentialapplication of Desmos chinensis extracts for controlling rice sheath blight. The dichloromethane extractfrom D. chinensis demonstrated high antifungal activity against Rhizoctonia solani. The extract was shownto have anti-R. solani activity with minimum inhibitory concentration (MIC) and minimum fungicidalconcentration values ranging from 31.2 to 62.5 mgmL�1 and from 62.5 to 500 mgmL�1, respectively.Bioautography on thin-layer chromatography plates demonstrated antifungal activity of the extract withan Rf value of 0.33. A total of 7 compounds, 2 benzoate esters (benzyl benzoate and benzyl2-hydroxybenzoate), 2 sesquiterpenoids (a-eudesmol and b-eudesmol), 1 aromatic alcohol (benzylalcohol), 1 aromatic ketone (acetophenone) and 1 diterpenoid (phytol), were detected and identifiedusing gas chromatographyemass spectrometry analysis. Electron micrographs confirmed the effects ofthe extract on morphological and ultrastructural alterations in the treated fungal cells. The micrographsof the mycelia treated with the extract at 4MIC illustrated aberrant morphology such as shrinkage, partialdistortion and globular structures of different sizes along the surface of the mycelia. Damagingmembranous structures including disruption of the cell membrane, partial loss of nuclear membranes,and depletion of hyphal cytoplasm and membranous organelles were observed. Foliar application ofD. chinensis extract at a concentration of 2 mgmL�1 on rice markedly decreased sheath blight severity. Itwas concluded that the application of D. chinensis extract can be used as a botanical fungicide to controlrice sheath blight.

Crown Copyright � 2012 Published by Elsevier Ltd. All rights reserved.

1. Introduction

Rice (Oryza sativa L.) is one of the most important staple cropsfor a large part of the world’s population, mainly in East, Southeastand South Asia (FAO, 2010). Losses due to diseases and pests are oneof the major constraints in rice production. Sheath blight of rice isa destructive disease in all crop-growing areas of the world. Thedisease is caused by a soil-borne fungal pathogen, Rhizoctonia solaniKühn (Teleomorph: Thanatephorus cucumeris (A.B. Frank) Donk)anastomosis group 1 subgroup 1A. The pathogen survives asmycelia or resistant structures known as sclerotia in plant debrisand on weeds in rice growing areas (Zachow et al., 2011). Highgenetic resistance is not available for sheath blight and the diseaseis currently managed through use of fungicides (Savary et al., 2012).

thikunchai).

012 Published by Elsevier Ltd. All

Several social and environmental problems occur around theworld due to the indiscriminate use of pesticides for controllingplant diseases. During the last few decades, there has been anincreasing interest in the study of secondary metabolites frommedicinal plants as antifungal agents for crop protection. Plantextracts and essential oils with antifungal activity against a varietyof fungi have been reported. Creosote bush and tarbush extractshad high antifungal activity against R. solani (Castillo et al., 2010).Clove extract completely inhibited the growth of R. solani,Rhizoctonia oryzae, Rhizoctonia oryzae-sativae and Sclerotiumhydrophilum (San Aye and Matsumoto, 2011). Siam weed extractwas found to be effective in reducingseverity of blast, brown spotand bacterial leaf blight disease of rice (Khoa et al., 2011). Thesemetabolites including alkaloids, glucosinolates, indole, phenolics,phenylpropanoids, saponins, stilbenes and terpenoids have beenreported to exhibit antifungal properties (Dixon, 2001; Lanzottiet al., 2012).

rights reserved.

P. Plodpai et al. / Crop Protection 43 (2013) 65e7166

Desmos chinensis Lour. is distributed in southern Asiancountries and used as folk medicines for treatment of malaria(Kakeya et al., 1993), parturition, vertigo (Rahman et al., 2003),pyretic, dysentery (Bunyapraphatsara and Chokchaichareonporn,2000), antirheumatic, antispasmodic and analgesic (Loi, 2001).Organic extracts of D. chinensis have been found to be effectiveagainst dermatophytic fungi (Kummee and Intaraksa, 2008).Previous chemical studies on D. chinensis have revealed in thisspecies presence of benzoic acid derivatives (Wu et al., 2000),benzoate ester derivatives (Van Kiem et al., 2005), C-benzylatedchalcones (Rahman et al., 2003), biflavones (Rittiwong et al.,2011), flavones, and oxoaporphine alkaloids (Lui et al., 2004).Since these extracts can be active against fungal and bacterialpathogens, easily biodegradable to non-toxic products, andpotentially suitable for use in integrated pest managementprograms, they could lead to development of new classes ofpossible safer disease control agents (Peshin et al., 2009). In thecase of natural fungicides, low toxicity and high efficiency arenecessary. The objectives of this research were to determine theantifungal properties and characterize the active compounds ofD. chinensis extracts. In addition, the effects on structural alter-ations on hyphal morphology and ultrastructural changes wereexamined using scanning electron microscopy and transmissionelectron microscopy.

2. Materials and methods

2.1. Plant pathogenic fungus

A multinucleate and virulent isolate of R. solani AG-1 IA wasobtained from Natural Biological Control Research Center, Prince ofSongkla University, Songkhla, Thailand. This isolate has beenextensively used in previous studies (Kanjanamaneesathian et al.,2007; Wiwattanapatapee et al., 2007; Chumthong et al., 2008).The stock culture was maintained on PDA at 4 �C.

2.2. Plant material

D. chinensis leaves and stem barks were collected from Songkhlaprovince in the southern part of Thailand in October 2009. Identi-fication was made by Assoc. Prof. Dr. Kitichate Sridith and a spec-imen (No. 0013593) was deposited at PSU Herbarium, Departmentof Biology, Faculty of Science, Prince of Songkla University.

2.3. Media and chemical reagents

Potato dextrose agar (PDA, Difco Laboratories, USA), RoswellPark Memorial Institute 1640 medium with L-glutamine andwithout bicarbonate (RPMI 1640, Sigma-Aldrich, USA), 3-(N-mor-pholino)-propanesulfonic acid (Applichem, Germany), propicona-zole (Irvita plant protection, Netherlands), dimethyl sulphoxide(DMSO, Merck, Germany) glutaraldehyde (Merck), dichloro-methane, ethyl acetate, ethanol, hexane and methanol (J.T. Baker,USA) were used.

2.4. Preparation of D. chinensis extracts

Samples were washed with distilled water, dried in an oven at60 �C, for 2 days, and then crushed using an electrical blender.Powdered samples were extracted in dichloromethane (1:5 (w/v))at room temperature for 7 days and then filtered throughWhatmanNo. 3 filter paper (Whatman Int. Ltd., UK). The plant materials wereextracted three times and the combined filtrates were evaporatedunder reduced pressure in a rotary evaporator (BUCHI Rotavapor R-114, Switzerland) and stored at 4 �C.

2.5. Determination of minimum inhibitory concentration andminimum fungicidal concentration

The broth microdilution method (CLSI, 2008) was used todetermine the minimum inhibitory concentration (MIC) ofD. chinensis extracts. The extract was dissolved in DMSO and dilutedin RPMI-1640 medium with L-glutamine and without bicarbonate,buffered to pH 7.0 with 0.165 M 3-(N-morpholino)-propanesulfonicacid and supplemented with 2% glucose. The extract (50 mL) wasdiluted to final concentrations ranging from 1.9 to 1,000 mgmL�1 in96-well microtitre plates. The same volume of mycelial suspensioncontaining approximately 104 CFUmL�1 was inoculated and incu-bated at 25 �C for 48 h. In addition, a reference antifungalcompound, propiconazole, was used as a positive control and 1%DMSO was used as a negative control. MIC was observed at least induplicate as the lowest concentration that completely inhibitedvisible growth. To determine a minimum fungicidal concentration(MFC), 100 mL from each of the wells at or above the MIC was platedon PDA and incubated at 25 �C for 72 h. MFC was defined as thelowest concentration at which no colonies were detected on theagar plate.

2.6. Growth inhibition assay

The anti-R. solani activities of the extracts were performed byinhibition of mycelial growth test (Rios et al., 1988). The extractwas dissolved in DMSO and 200 ml was added to PDA to achievea final concentration at 200 mgmL�1. A 5 mm agar disc con-taining mycelia was transferred to the center of the PDA platecontaining the extract. Plates were incubated at 25 �C for 3 days.Propiconazole was used as a positive control and 1% DMSO wasused as a negative control. Radial growth was assessed bymeasuring the distance from the edge of the inoculum plug tothe advancing margin of the colony. Growth inhibition oftreatment against control was calculated according to Gamlielet al. 1989. Each experiment was conducted twice in fourreplicates.

Percentage growth inhibition ¼ 100���

R2

r2

�� 100

�

where R and r represent the radius of fungus colony in the treatedand control plates, respectively.

2.7. Thin-layer chromatography-direct bioautography

Thin-layer chromatography (TLC)-direct bioautographic methodcombined with gas chromatography-mass spectrometry (GCeMS)analysis was applied to identify and localize active compounds inthe extract (Scher et al., 2004). The extract was dissolved in hexaneat a concentration of 10 mgmL�1. Ten microliters of this solutionwere applied on silica gel TLC plates (silica gel 60 F254 (0.2 mmthick); Merck). TLC plate loaded with the extract was developed ina hexane:ethyl acetate (8:1, v/v) solvent system and thoroughlydried for complete removal of the solvents. Overlay media wasdistributed over the developed TLC plates. After solidification of themedia, the chromatograms were inoculated with four mycelial agardiscs and incubated at 25 �C for 48 h. A silica gel band, whichshowed antifungal activity against R. solani, was collected byscraping off the band and then extracted with methanol andcentrifuge at 10,000 rpm for 5 min. The antifungal extract wasconcentrated to dryness and analyzed using GCeMS with a Hew-lett-Packard HP 5890 series II gas chromatograph (Agilent Tech-nologies, US).

P. Plodpai et al. / Crop Protection 43 (2013) 65e71 67

2.8. Effects of D. chinensis extract on hyphal morphology

Effects of the extract on hyphal morphology of R. solani wereobserved using scan electron microscopy (SEM). Fungal cells werecultured at 25 �C for 24 h in RPMI 1640 medium in the presence orabsence of the extract at 4MIC concentration. The mycelia treatedwith 1% DMSO were used as control. These mycelial plugs wereprepared for both SEM and TEM. Specimens were fixed in 2.5%glutaraldehyde in 0.1 M phosphate buffer, pH 7.3 for 2 h at roomtemperature. After being washed with the buffer, the specimenswere post-fixed in 1% osmium tetroxide in the same buffer for 1 hat room temperature. The specimens were dehydrated in a series ofgraded ethanol series (50, 60, 70, 80, 90 and 100%) for a period of15 min in each series. Fixed samples were critical point dried undercarbon dioxide and sputter-coated with gold. Morphologicalchanges of the fungal cells were observed by SEM Quanta 400, FEIat 10 kV.

2.9. Effects of D. chinensis extract on hyphal ultrastructure

For transmission electron microscopy (TEM) preparation, thesamples were fixed in phosphate-buffered 2.5% glutaraldehyde,post-fixed in phosphate-buffered 1% osmium tetroxide and dehy-

Disease index ¼� PðThe number of diseased plants in the index� Disease indexÞðTotal number of plants inverstigated� The highest disease indexÞ

�� 100%

drated in a graded series of ethanol following the procedurementioned in Section 2.6. TEM samples were infiltrated with

Control efficacy ¼�Disease severity of control� Disease severity of treatedgroup

Disease severity of control

�� 100%

ethanoleresin mixtures and embedded in pure epoxy resin andpolymerized in an oven at 80 �C for 24 h. The ultrathin sectionswereobtained inultramicrotome, contrastedwith uranyl acetate and leadcitrate and observed using TEM 100 CX-II, JEOL (Japan) at 80 kV.

Table 1Minimum inhibition concentration (MIC) and minimum fungicidal concentration(MFC) of Desmos chinensis extracts (MIC/MFC, mgmL�1) against Rhizoctonia solaniusing the broth microdilution method.

Compound MIC (mgmL�1) MFC (mgmL�1)

Leaf extract 31.2 62.5Stem bark extract 62.5 500.0Propiconazole 1.9 1.9

2.10. Effects of D. chinensis extract on the incidence of sheath blightdisease

Efficacy of D. chinensis extract against sheath blight was con-ducted under pot culture conditions. Rice seeds cultivar Phitsanu-lok 2 were surface sterilized in 10% clorox for 1.5 min, rinsed threetimes in sterile distilled water, and dipped in sterile distilled waterfor 1 day for imbibition prior to the germination trial. Germinatedseedlings were grown in a nursery bed. Rice seedlings wereremoved after 25 days, then transplanted at the rate of four seed-lings per tiller and five tillers per pot (38 cm in diameter containing10 kg of clay soil). Nitrogen, phosphorus and potassium fertilizerswere applied in the form of urea, single super phosphate, andmuriate of potash at 1.1, 0.93 and 0.2 g pot�1, respectively. Thesewere applied at 50% as basal (before transplanting) and remaining50% in two split applications at 25 and 45 days after trans-plantation. The pots were placed in a completely randomizeddesign with five treatments, four replicates and each replicatecontaining 20 rice plants. Rice plants at late tillering stage were

inoculated with R. solani by placing a mycelial ball below the leafsheath as previously described (Park et al., 2008). The inoculatedsheath was covered immediately with aluminum foil. When typicallesions appeared after 3 days, the aluminum foil was removed.

D. chinensis extract was dissolved in DMSO and diluted in waterto achieve final concentration at 0.5, 1 and 2 mgmL�1. A commer-cial fungicide, propiconazole, was used to control sheath blight atrecommended concentrations of 1.25 mgmL�1. All the treatmentswere applied as a finemist to the upper leaf blades until runoff withan aerosol sprayer at 7, 14 and 21 days after inoculation.

Visual disease assessment was determined 7 days after the thirdtreatments application. Disease severity was scored as grades 0e9according to the standard evaluation system of International RiceResearch Institute (IRRI, 2002). Severity of sheath blight was scoredwith a scale of 0e5 based on relative lesion height on thewhole plant(IRRI, 2002) as follows: 0¼ no infection; 1¼ lesions limited to thelower 20% of plant height; 2¼ lesions limited to the lower 20e30% ofplant height; 3¼ lesions limited to the lower 31e45% of plant height;4¼ lesions limited to the lower 46e65% of plant height; and5¼ lesions on theupper 65%of plant height. The relative lesionheightis the average vertical height of theuppermost lesion on leaf or sheathexpressed as a percentage of the average plant height. Based on thegrades, disease index of sheath blight was calculated by the formula:

Control efficacy of the test materials on sheath blight diseasewas evaluated with control value calculated by the formula:

2.11. Statistical analysis

All datawere analyzed by one-way analysis of variance (ANOVA)and comparison of means using the Duncan’s Multiple Range Testat the level P< 0.05. The statistical analysis was performed usingstatistical package for the social sciences 15.0 software forWindows (SPSS Inc., Chicago, IL, USA).

3. Results and discussion

3.1. Antifungal susceptibility testing

Significant antifungal effects of the dichloromethane extractsfrom D. chinensis leaves and stem barks expressed as MIC and MFCvalues against R. solani are presented in Table 1. The resultsdemonstrated that D. chinensis extracts exhibited antifungal

Table 2Antifungal activity of Desmos chinensis extracts against Rhizoctoniasolani using inhibition of mycelial growth test.

Compounds Mean of percentage mycelialinhibition� standard error

Leaf extract 95.3� 0.2b

Stem bark extract 88.0� 0.5c

Propiconazole 100.0� 0.0a

1% DMSO 00.0� 0.0d

The results are means� standard errors of four replications. Meanswithin a column indicated by the same letter were not significantlydifferent according to Duncan’s multiple range test at the levelP< 0.05.

P. Plodpai et al. / Crop Protection 43 (2013) 65e7168

activity against R. solani with MIC values ranging from 31.2 to62.5 mgmL�1. Interestingly, D. chinensis leaf extract has higheractivity than stem barks with MIC at 31.2 mgmL�1 and MFC at62.5 mgmL�1. Propiconazole exhibited fungicidal ability with MICand MFC at 1.9 mgmL�1. However, the MIC and MFC of the standardfungicide were significantly lower than that of the extracts. Thefindings are consistent with data obtained from previous studies(Kummee and Intaraksa, 2008; Tuntipaleepun et al., 2012).

3.2. Growth inhibition assay

The results, as shown in Table 2, indicated that the dichloro-methane extracts from D. chinensis leaves and stem barks werehighly effective against R. solani with 95.3 and 88.0% inhibition,respectively. The suppressive effect of the extracts on myceliumgrowth was significantly less than that of the standard fungicide.Despite of the fact that the efficacy of the extract was significantlylower than that of the standard chemical fungicide, we decided touse the extract for the further experiment since its MIC and MFCwere better than previously reported extracts from other plantextracts.

3.3. Thin-layer chromatography-direct bioautography

As shown in Fig. 1A, clear inhibition zone was observed againstR. solani with an Rf value of 0.33, suggesting that the substances

Fig. 1. (A) Bioautography on silica TLC plates demonstrated antifungal activity of Desmos chigrowth. TLC plate loaded with the extract developed in a hexane:ethyl acetate (8:1, v/v) solve25 �C for 48 h. (B) Main compounds identified in Desmos chinensis:. (1) acetophenone; (2) bbenzyl 2-hydroxybenzoate.

responsible for anti-R. solani activity are non-polar. Activecompounds, including two benzoate esters (benzyl benzoate andbenzyl 2-hydroxybenzoate), two sesquiterpenoids (a-eudesmoland b-eudesmol), an aromatic alcohol (benzyl alcohol), an aromaticketone (acetophenone) and a diterpenoid (phytol), were identifiedby mass spectral library search (Fig. 1B). Previous chemical inves-tigations have shown that D. chinensis possess a number of chem-ically and biologically interesting compounds which includebenzoic acid derivatives (Wu et al., 2000), benzoate ester deriva-tives (Tuntipaleepun et al. 2012) and biflavones (Rittiwong et al.,2011). The presence of benzyl benzoate and benzyl 2-hydroxybenzoate supported the antifungal properties ascribed to

nensis extract (10 mgml�1). Clear zones on bioautograms indicated inhibition of fungalnt system. The chromatogramwas inoculated with mycelia of R. solani and incubated atenzyl alcohol; (3) a-eudesmol; (4) b-eudesmol; (5) phytol; (6) benzyl benzoate and (7)

P. Plodpai et al. / Crop Protection 43 (2013) 65e71 69

the plant by folk medical tradition in a previous pharmacologicalinvestigation. Among them, benzoate esters are usually employedas fungicides because they are able to affect the sporulation processand mycelial growth (Kim et al., 2010). Inhibition of benzoate para-hydroxylase (Podobnik et al., 2008), futile proton pumping thatdepletes ATP stores (Lambert and Stratford,1999) and alterations inmembrane permeability (Holyoak et al., 1999) by these compoundshave been reported as the main mechanism of action.

3.4. Effects of D. chinensis extract on hyphal morphology

Scanning electron micrographs clearly demonstrated that thegrowth of R. solani in media containing the extract caused profoundchanges in cell morphology. R. solani mycelium grown in RPMI-1640 medium as control displayed the characteristic morphologywith typical tapered apices and a smooth surface (Fig. 2A and B).After treatment with the extract at 125 mgmL�1 (Fig. 2C and D), theprominent morphological changes appeared were rough cells withwrinkle cell surfaces and globular structures of different sizes alongthe surface of the mycelia. Mycelia of R. solani were completelycollapsed and folding with the formation of short branches andundifferentiated tips. The morphological changes might result fromthe destruction of organelles in the endomembranes system.Alterations and damage on vegetative hyphae or conidia have beenpreviously described by many workers (Hashem, 2011; Khan andAhmad, 2011). However, SEM observation on R. solani has not yet

Fig. 2. Scan electron microscopy SEM micrographs of Rhizoctonia solani hyphae. Control witminimum inhibition concentration 125 mgmL�1 shows hyphal tips appearing shriveled and

reported. The impacts of the extract on fungal structures have beensuggested to be due to alterations in membrane permeability byhigh concentrations of benzoate ester derivatives and biflavones(Rittiwong et al., 2011).

3.5. Effects of D. chinensis extract on hyphal ultrastructure

Transmission electron microscopy observation of untreatedR. solani hyphae revealed typical eukaryotic cytoplasmic compo-nent including numerous ribosomes, nucleus, mitochondria andvacuoles in the cytoplasm enclosed by an electron-dense cell wall.The cell membrane displayed as a sharp, electron-dense phos-pholipid bilayer closely attached to the cell wall (Fig. 3A and B).Mitochondria showed well-developed cristae projecting into thematrices. When the fungal cells were exposed to the extract at125 mgmL�1, it presented cytoplasmic vacuolation, folding andbreaking down of the cell membrane at various sites and detach-ment from the cell wall (Fig. 3C and D). Complete autolysis, disor-ganization and depletion of hyphal cytoplasm and membranousorganelles including nuclei, mitochondria and endoplasmic retic-ulum seemed to be responsible for cell death. The ability of theextract to induce rearrangement of intracellular membranes,breaks in the nuclear envelope and intermingling of nuclear andcytoplasmic contents may be a result of benzoate esters and ben-zoic acid derivatives that were reported to be present in the leaves(Rittiwong et al., 2011).

h 1% DMSO, showing healthy hyphae (A and B). The hyphae treated with the extract atglobular structures of various sizes along the surface of the mycelia (C and D).

Fig. 3. Transmission electron microscopy micrographs of Rhizoctonia solani hyphae. Control with 1% DMSO, hyphae revealed typical eukaryotic cytoplasmic component includingnumerous ribosomes, nucleus, mitochondria and vacuoles in the cytoplasm enclosed by an electron-dense cell wall (A and B). At minimum inhibition concentration, 125 mgmL�1,the cell membrane of the treated R. solani which was attached to the cell wall became separated from the wall after treatment with the extract. Complete autolysis, disorganization,and depletion of hyphal cytoplasm and membranous organelles seemed to be responsible for cell death (C and D). CW¼ cell wall; CM¼ cell membrane; N¼ nucleus;M¼mitochondrion; R¼ ribosome. Magnifications were as follows: A and C, �4000; B and D, �10,000.

Table 3Efficiency of Desmos chinensis extract for controlling rice sheath blight under pot culture conditions.

Treatment Sheath blight score (1e9 scale) Disease index (%) Suppression efficacy (%)

Untreated control 7.3� 0.0e 80.8� 0.5e d

Desmos chinensis extract (mgmL�1)0.5 6.2� 0.1d 68.3� 0.7d 15.5� 0.9d

1.0 5.6� 0.1c 61.7� 0.7c 23.7� 0.9c

2.0 5.1� 0.0b 56.7� 0.3b 29.9� 0.4b

Propiconazole(1.25 mgmL-1)

4.6� 0.0a 51.5� 0.3a 36.3� 0.4a

The results are means� standard errors of four replications. Means within a column indicated by the same letter were not significantly different according to Duncan’smultiple range test at the level P< 0.05.Severity of sheath blight was scored with a scale of 0e5 based on relative lesion height on the whole plant as follows: 0¼ no infection; 1¼ lesions limited to the lower 20% ofplant height; 2¼ lesions limited to the lower 20e30% of plant height; 3¼ lesions limited to the lower 31e45% of plant height; 4¼ lesions limited to the lower 46e65% of

plant height; and 5¼ lesions on the upper 65% of plant height. Disease index ¼� PðThe number of diseased plants in the index� Disease indexÞðTotal number of plants inverstigated� The highest disease indexÞ

�� 100%.

Suppression efficacy ¼�Disease severity of control� Disease severity of treated group

Disease severity of control

�� 100%.

P. Plodpai et al. / Crop Protection 43 (2013) 65e7170

3.6. Efficacy of D. chinensis extract on sheath blight severity

The data, as shown in Table 3, indicated that foliar application ofthe extract markedly reduced the incidence of sheath blight diseasecompared to untreated. Sheath blight severity in rice was signifi-cantly decreased by propiconazole at 1.25 mgmL�1 followed by theextract at concentration of 2, 1 and 0.5 mgmL�1 as compared tocontrol. The disease severity indexes in control plots were 80.8%,while application of the extract at a concentration of 2 reduced theseverity to 29.9%. Various other plant species have been used tocontrol the pathogen in the field (Nisha et al., 2012). Fresh leafextract of Chromolaena odorata reduced lesion length of sheathblight but disease development was not prevented (Khoa et al.,2011). Foliar application methanol leaf extracts of Datura metel

was able to control sheath blight and bacterial blight diseasesthrough a direct antimicrobial effect of this extract as well as byinduced resistance (Kagale et al., 2004). Investigations on themechanisms of disease suppression by plant extracts have sug-gested that the active principles present in them may either act onthe pathogen directly (Abdel-Monaim et al., 2011), or inducesystemic resistance in host plants resulting in reduction of diseasedevelopment (Kagale et al., 2011).

4. Conclusions

This study indicated that D. chinensis extracts possessed markedantifungal property against R. solani. The dichloromethane extractsprimarily affected fungal cell permeability through direct

P. Plodpai et al. / Crop Protection 43 (2013) 65e71 71

interaction with the cell membrane. A change in cell permeabilitymight have resulted in an imbalance in intracellular osmoticpressure, subsequent disruption of intracellular organelles, leakageof cytoplasmic contents and finally cell death. Applications ofD. chinensis formulations as an alternative fungal control will befurther investigated in order to minimize side-effects produced bythe use of chemicals in crop protection.

Acknowledgments

This work was supported by the Thailand Research Fund,Natural Products Research Center, and PSU Ph.D. Scholarship,Prince of Songkla University.

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