Analysis of Antifungal Components in the Galls of Melaphis chinensis and Their Effects on Control of Anthracnose Disease of Chinese Cabbage Caused by Colletotrichum higginsianum

1Department of Biotechnology, National Formosa University, Yunlin 632, Taiwan 2Floriculture Research Center, Taiwan Agricultural Research Institute, Council of Agriculture, Executive Yuan, Yunlin 646, Taiwan 3Department of Plant Pathology, National Chung Hsing University, Taichung 402, Taiwan 4Plant Pathology Division, Taiwan Agricultural Research Institute, Council of Agriculture, Executive Yuan, Wufeng, Taichung 413, Taiwan


Introduction
There are numerous reports indicating that tissues of some plant species contain antifungal substances, including rhizomes of Curcuma longa [1], seeds of Cassia tora [2], stem/leaves and flowers of Lavandula stoechas [3], and others.For example, seeds of mustard (Brassica juncea cv.Bau Sin) are rich in glucosinolate and enzymatic hydrolysis of this compound resulted in the release of allyl isothiocyanate that is highly toxic to Rhizoctonia solani Kühn AG-4, causal agent of root rot of cabbage [4].Some plant species with antifungal properties are also used as medicinal plants.For example, galls of Melaphis chinensis [5,6] and leaves of Aloe vera [7] contained toxic substances against plant pathogenic fungi.

Journal of Chemistry
Water-soluble extracts of clove completely inhibited the conidial germination and mycelial growth of C. higginsianum at the concentration of 1% (w/v).In addition, clove oil and eugenol were equally effective in reducing disease severity of anthracnose caused by this pathogen in greenhouse [15].Although the antifungal activities of various plants were extensively reported, there were relatively few studies regarding the antifungal principles in the plant extracts.
Colletotrichum species are important fungal pathogens causing anthracnose disease of numerous economically important crops, including legumes, ornamentals, vegetables, and fruit trees [16][17][18][19][20][21][22] and thus are responsible for severe yield losses of cabbage crops in commercial fields in Taiwan [23].Although these diseases could be successfully controlled by the synthetic chemical fungicides, the utilization of synthetic fungicides led to the development of resistance and environment pollution.The biological control of plant diseases which is recognized as use of metabolites from the natural source is an eco-friendly resolution [24].In our preliminary experimental results (Table 1), forty herbal extracts were examined for their antifungal activity against C. higginsianum PA-01.Most of them displayed inhibition of the fungus and among the tested methanol extracts, four species of medicinal plants, Melaphis chinensis, Eugenia caryophyllata, Polygonum cuspidatum, and Rheum officinale, exhibited the inhibitory percentages between 80.64 ± 3.16 and 91.92 ± 3.00% at the tested concentration (1250 g/mL).The experimental data indicated that these extracts possessed antifungal activity to causal agent of cabbage anthracnose C. higginsianum.However, the chemical nature of the antifungal substances in these Chinese herbs remains unknown.Therefore, the objectives of this study were to identify the compounds and their antifungal activity in four species of medicinal plants.In addition, the indicator compounds were used as standards to quantitatively analyze these medicinal plants with the aid of high performance liquid chromatography (HPLC) and the validation examinations were performed to confirm that these methods were precise and reliable for quality evaluation.Thus they could be utilized to control the quality of herbal preparations to ensure their antifungal activities.Moreover, their effects of controlling anthracnose disease of Chinese cabbage caused by Colletotrichum higginsianum PA-01 in growth chamber were also examined.

Materials and Methods
2.1.General Procedure.All the solvents including the HPLCgrade methanol were purchased from Merck KGaA (Darmstadt, Germany).The chemical structures of the indicator compounds were identified by comparison of their spectroscopic and physical data with those reported in the literature.Their purities were better than 99.0% as determined by HPLC.Plant materials were extracted using a Major Science LM-570R shaking incubator.High performance liquid chromatography (HPLC) was performed on a Shimadzu LC-20ATseries pumping system equipped with a Shimadzu SPD-20AUV-Vis detector, a Gemini 5u C18 column (4.6 mm × 250 mm, 5 m), and a SIL-10AF autosampling system.

Fungal Pathogen and Plant Materials.
Two isolates (PA-01 and PA-19) of C. higginsianum were used in this study.They were isolated from diseased leaves of cabbage (Brassica rapa L. Chinese group) grown in Yunlin, Taiwan.The cultures were maintained on potato dextrose agar (PDA, Difco, USA).Dry powders of all the examined medicinal herbs were purchased from herbal stores in Yunlin, Taiwan.All the purchased materials for the experiments were authenticated by Dr. T. F. Hsieh and the voucher specimens (PCKuo TFHsieh 201001-201040) were deposited in the herbarium of Department of Biotechnology, National Formosa University, Yunlin, Taiwan.Seeds of Chinese cabbage (B.rapa L.) were put on a number 1 filter paper (9 cm in diameter, Toyo Roshi Co., Japan) moistened in water and kept in a Petri dish at room temperature (22-25 ∘ C) for 1 day.The germinated seeds were sown in peat moss in plastic pots, 128 cells/tray, and 1 seed/cell.After one week, individual seedlings were transplanted to plastic pots (18 cm in diameter) filled with Stender peat substrates (Stender AG, Germany), 3 plants/pot, and kept in a greenhouse for four weeks with daily watering.

Effect of the Methanol Extracts on Conidial Germination of C. higginsianum.
The methanol extracts were tested for inhibition of conidial germination of C. higginsianum PA-01 according to the method of Lee and Dean [25].The isolate was grown on oatmeal agar (50 g/L) at 25 ∘ C under continuous fluorescent light.Conidia were harvested from 7to 10-day-old cultures and the solution was filtered to collect conidial suspension.Ten microliters of conidial suspension (∼10 5 conidia/mL) was mixed with ten microliters of different extracts.The cultures were placed in a moistened plastic box, incubated at 25 ∘ C for 24 h, and examined for germination of conidia under a compound microscope.There were six replicates for each sample (100 conidia/replicate).Sterile distilled water was used as negative control and azoxystrobin was used as positive control.Inhibition rate of conidia for each treatment was calculated by extract was partitioned between ethyl acetate and water to afford ethyl acetate solubles (ECE, 5.5 g) and water extracts (ECW, 1.5 g), respectively.The roots of P. cuspidatum (100.0 g) were extracted with methanol under reflux (0.3 L × 5 × 8 h), and the crude extracts were concentrated to give a brown syrup (PC, 8.0 g).The crude extract was partitioned between chloroform and water to afford chloroform solubles (PCC, 1.7 g) and water extracts (PCW, 6.3 g), respectively.The roots of R. officinale (100.0 g) were extracted with methanol under reflux (0.3 L × 5 × 8 h), and the crude extracts were concentrated to give a brown syrup (RO, 27.0 g).The crude extract was partitioned between chloroform and water to afford chloroform solubles (ROC, 2.8 g) and water extracts (ROW, 24.2 g), respectively.

Purification and Identification of Indicator Compounds.
The methods for purification and identification of indicator compounds in the four medicinal plants were described as follows.
(I) M. chinensis.The ethyl acetate soluble fraction (MCRE, 40.0 g) of the crude extract was applied to a silica gel column and then eluted with chloroform and step gradient of ethyl acetate (10 : 1 to 1 : 1, v/v) to yield 9 fractions.Fraction 3 was subjected to silica gel column chromatography eluted with n-hexane and acetone (10 : 1, v/v) to yield methyl gallate (2) (10.5 g).Fraction 8 was further resolved on a silica gel column eluted with chloroform and acetone (5 : 1) to give gallic acid (1) (2.3 g).
(II) E. caryophyllata.The ethyl acetate soluble fraction (ECE, 5.5 g) of the crude extract was purified with silica gel column chromatography and eluted with n-hexane and step gradient of ethyl acetate (20 : 1 to 1 : 1, v/v) to yield 5 fractions.Fraction 2 was further purified on a silica gel column eluted with chloroform and ethyl acetate (20 : 1, v/v) to give eugenol (3) (250.0 mg).
(III) P. cuspidatum and R. officinale.For P. cuspidatum, the chloroform soluble fraction (PCC, 1.7 g) of the crude extract was purified with silica gel column chromatography and eluted with chloroform and step gradient of ethyl acetate (50 : 1 to 1 : 1, v/v) to yield 9 fractions.Fraction 2 was further recrystallized with chloroform and ethyl acetate to afford physcion (6) (25.0 mg).Fraction 6 was further resolved on a silica gel column eluted with chloroform and step gradient of methanol (100 : 1 to 1 : 1, v/v) to yield emodin (4) (30.0 mg).For R. officinale, the chloroform soluble fraction (ROC, 2.8 g) of the crude extract was purified with silica gel column chromatography and eluted with chloroform and step gradient of methanol (300 : 1 to 1 : 1, v/v) to afford 11 fractions.Fraction 2 was further purified with the assistance of silica gel column chromatography eluted with n-hexane and acetone (100 : 1, v/v) to give chrysophanol (5) (28.0 mg).

Preparation of Standard Solutions, Calibration Curves, and
Validation of the Analytical Methods.The standard solutions and calibration curves for the six indicator compounds were prepared according to the methods reported in the literature [29].The reproducibility and precision of detection were measured by repeatedly injecting a ready-made sample pool and expressed as the relative standard deviation of the results.
To determine the variance of samples within a day, the same samples were tested at different times within the day.The variance between days was determined by assaying the spiked samples over three consecutive days at the same time each day.The limit of detection (LOD) was determined as the lowest detectable concentration with acceptable accuracy and precision and three times above the noise level (S/N ≥ 3).The recovery of the indicator compounds was evaluated using three different concentrations covering the linear range of the standard curve and the peak heights were compared to the standard compounds to calculate the recovery data.were inoculated on each plant at 8 mL/plant and 10 5 conidia/mL, using a compressed air-sprayer (SIL-AIR, Werther International, Italy).All pots were placed in moist plastic bags and kept in a growth chamber at 24 ∘ C under 12 h diurnal illumination.The plastic bags were removed after one-day incubation and the plants were examined for lesion number and infection area in 3 cm diameter of leaf spot at 5, 7, and 9 days after inoculation.

Statistical Analysis.
Data collected from all the experiments in this study were analyzed for statistical significance using analysis of variance (ANOVA).Means of treatments in each experiment were separated using Duncan's multiple range tests.The analytical results are expressed as mean ± standard deviation (SD).Relative standard deviations (RSDs) were calculated from those values.In addition, the mean values of lesion number and lesion area on infection leaves were analyzed by the least significant difference (LSD) test.Although the synthetic fungicides successfully controlled the plant diseases sometimes, they also contributed to increasing the population of fungicideresistant pathogens [30].Natural plant metabolites are generally considered as safe to the humans and environment since these chemical compounds are easily decomposed in the soil and would not exhibit long-term effects to the environment [31].Thus more and more reports were focused on the development of new plant-derived pesticide preparations recently [1,4,15,32,33], but comparatively few studies related to the antifungal principles in the bioactive extracts were completed.These new preparations would be hopeful to reduce the damage caused by traditional synthetic fungicides and in the meanwhile to suppress the disease development effectively.Detailed chemical analysis of the constituents in the plant extracts is helpful to confirm the active compounds and control the quality of the plant-derived pesticide preparations.

Identification of Indicator Compounds in the Medicinal
Plant Extracts.The indicator compounds (Figure 1), including gallic acid (1) [34] and methyl gallate (2) [35] from the galls of M. chinensis, eugenol (3) [36] from buds of E. caryophyllata, emodin (4) [37] and physcion (6) [38] from the roots of P. cuspidatum, and chrysophanol (5) [39] from the roots of R. officinale, were purified and characterized by comparison of their spectral and physical data with those reported in the literature.The purity of all the indicator compounds except physcion (6) as determined by HPLC was better than 99.0%.

Optimization of the HPLC Condition and Method Validation.
The optimized HPLC analytical conditions for the medicinal plant extracts were designed as displayed in the experimental section.The calibration curve parameters and limits of detection (LOD) for the indicator compounds were displayed in Table 2.The precision of the HPLC method developed was evaluated through the intraday and interday experiments.Among the linear ranges, the RSDs for all the indicator compounds of the intraday and interday precisions were found to be less than 1.62 and 2.61%, respectively (Table 2).The recovery of the indicator compounds was determined by the addition of a sample with known concentration to the standard solution, and the mean recovery rate was found to be in the ranges from 81.12 to 126.33%  4) Chrysophanol ( 5) Physcion ( 6) with satisfactory RSDs in the ranges between 0.09 and 3.26% (Table 2).
In the present study, the indicator compounds in M. chinensis, E. caryophyllata, C. cassia, P. cuspidatum, and R. officinale had been extracted and purified.The indicator compounds were further used as standards to quantitatively analyze these traditional Chinese medicines with the aid of HPLC and the validation examinations were carried out to confirm that these methods were precise and reliable for quality evaluation.In the development of the HPLC method for the quantitative determination of indicator compounds, several solvent systems and separation columns were evaluated and compared.Detection wavelength was also optimized in this work.The maximum number and the heights of the peaks of the constituents were obtained and the baseline of chromatogram was stable.The reproducibility of the analytical method was performed and the results showed that it was satisfactory with the RSDs below 3.0% for any of the indicator compounds (data not shown).The precision and recovery tests all displayed that the established HPLC chromatographic methods were valid for the quantitative determination of the indicator compounds and also convenient and feasible as tools for species authentication and quality assessment of the herbal raw materials.

Quantitative Determination of Indicator Compounds in
the Medicinal Plant Extracts.The developed HPLC chromatographic analytical methods were applied to assess the contents of the indicator compounds in the extracts of corresponding plant materials and the data were displayed in Table 3.The contents of methyl gallate (2) and eugenol (3) in the ethyl acetate soluble fractions of methanol extracts of M. chinensis and E. caryophyllata, respectively, were more than 30% and they indicated that these constituents were the major component in the plant extracts.In contrast, emodin (4), chrysophanol (5), and physcion (6) were less than 5% in the methanol extracts of P. cuspidatum and R. officinale.The reproducibility of the analytical results was satisfactory with the RSDs below 3.53% for all the examined indicator compounds.2), respectively, compared to the reference synthetic pesticide azoxystrobin (IC 50 0.5 and 0.4 g/mL against C. higginsianum PA-01 and PA-19, resp.).Among the examined samples, most of them displayed significant inhibition of the conidial germination of the pathogen and this indicated that these plant-derived pesticide preparations were promising.

Effect of Gallic Acid and Methyl Gallate on Control of Anthracnose Disease of Chinese Cabbage Caused by C.
higginsianum PA-01 in Growth Chamber.Suppression of Chinese cabbage anthracnose by indicator compounds, gallic acid, and methyl gallate was dependent on the concentration, where lesion area (%) and lesion number per 3 cm in diameter of infected leaf were decreased by increasing concentrations of each indicator compound (Table 6).In general, disease  suppression by methyl gallate was better than by gallic acid in the same concentration (Figure 3).For example, the lesion number was not a significant difference in treatment of methyl gallate with 14.9 at 500 g/mL and in treatment of gallic acid with 13.2 at 1000 g/mL 9 days after inoculation.Similarity, lesion area (%) was also not a significant difference in treatment of methyl gallate with 40.9 at 500 g/mL and in treatment of gallic acid with 38.4 at 1000 g/mL 9 days after inoculation (Table 6).It means that lower concentration of methyl gallate displayed better effects for disease control than higher concentration of gallic acid.The results would be valuable for the discovery of new plant-derived pesticide preparations.

Conclusion
The present investigation results indicate that the methanol extracts of M. chinensis, E. caryophyllata, P. cuspidatum, According to the experimental data in the present study, methyl gallate showed only 1/80 activity of the pesticide azoxystrobin; however, the herbal extracts would be safer and less dangerous to the ecosystem.These traditional Chinese medicines could be studied further for their cytotoxicity and synergistic effects of different combinations.It would be also potential to study the antifungal mechanism in the future.
, Partially Purified Fractions, and Indicator Compounds.The antifungal effects of the extracts and fractions on conidial germination of C. higginsianum PA-01 and PA-19 are displayed in Table 4.Most of the crude extracts and low polarity fractions displayed inhibitions of the conidial germination of the fungal pathogen.Among the tested samples, the ethyl acetate fraction of the methanol extracts of M. chinensis exhibited the most significant antifungal activities towards C. higginsianum PA-01 and PA-19 with the IC 50 values of 236.6 and 191.4 g/mL, respectively.The antifungal effects of the indicator compounds 1-6 and the reference compound azoxystrobin were displayed in Table 5. Compounds 1-5 all exhibited the inhibitory effects against C. higginsianum PA-01 with the IC 50 values less than 850.3 g/mL, and comparatively compounds 1-4 and 6 showed the significant inhibition of the conidial germination of C. higginsianum PA-19 with the IC 50 values ranging from 22.1 to 1259.0 g/mL, respectively.The major component methyl gallate (2) in the most active fraction (MCRE) displayed the most significant antifungal effects with the IC 50 values of 40.2 ± 0.01 (1.30) 3.94 ± 0.01 (0.32) N.D. 1.38 ± 0.01 (0.82) 1.79 ± 0.01 (0.55) N.D. a The contents of each compound were presented as mean ± S.D. (RSD %) (%, g/g sample).b Not determined.c Not detectable.

APFigure 2 :Figure 3 :
Figure 2: Effect of methyl gallate on inhibition of conidial germination of Colletotrichum higginsianum PA-19.(a) An irregular brown appressorium (AP) formed after conidial germination in distilled water (check); and (b) conidium (C) failed to germinate in the solution with 22.1 g/mL of methyl gallate (bar scale = 20 m).

Table 1 :
The preliminary antifungal screening of the different herbal extracts on conidial germination of Colletotrichum higginsianum PA-01.
3.1.Antifungal Activities of the Crude Extracts.The antifungal effects of the forty herbal extracts on conidial germination of C. higginsianum PA-01 are displayed in Table 1.Among the examined samples, four species of medicinal plants, including Melaphis chinensis, Eugenia caryophyllata, Polygonum cuspidatum, and Rheum officinale, displayed significant antifungal activity against C. higginsianum PA-01.Thus these four extracts were selected as the targets of developing new botanical pesticides.

Table 2 :
Calibration curve parameter, limits of detection (LOD), precision, and recovery for the indicator compounds.

Table 3 :
Contents of each indicator compound in the crudes and fractions of the examined herb extracts.

Table 4 :
Antifungal activity of crude extracts and fractions of Chinese medicinal herbs a .

Table 5 :
Antifungal activity of indicator compounds a .

Table 6 :
Effect of gallic acid and methyl gallate on control of anthracnose disease of Chinese cabbage caused by Colletotrichum higginsianum PA-01 in growth chamber.Days after inoculation.2LN:lesionnumberper 3 cm in diameter of infected leaves and LA (%): percentage of lesion area per 3 cm in diameter of infected leaves.3Data in each column with the same letter are significantly different according to LSD test in  = 0.05.