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Chemical composition and anti-fungal properties of the essential oils and crude extracts of Orthosiphon stamineus Benth
M. Amzad Hossain a, , Zhari Ismail b , Atiqur Rahman c,d , Sun Chul Kang c
a

Chemistry Division, Atomic Energy Centre, GPO Box 164, Ramna, Dhaka 1000, Bangladesh School of Pharmaceutical Sciences, University Sains Malaysis, 11800 Pulau Pinang, Malaysia c Department of Biotechnology, Daegu University, Kyoungsan, Kyoungbook 712-714, Republic of Korea d Department of Applied Chemistry and Chemical Technology, Islamic University, Kushtia 7003, Bangladesh
b

a r t i c l e
Article history:

i n f o

a b s t r a c t
The hydrodistilled essential leaves and stems oils of Orthosiphon stamineus Benth were analysed by GCMS/MS. Sixty nine compounds representing 97.6 and 97.4% of the total leaves and stems oils, respectively were identied, of which -humulene (14.2 and 18.4%), bourbonene (3.4 and 3.0%), -caryophyllene (24.0 and 35.1%), -elemene (11.1 and 8.5%), 1-octen-3-ol (8.2 and 7.0%),

Received 30 June 2007 Received in revised form 23 November 2007 Accepted 26 November 2007

-pinene (2.1 and 1.7%), caryophyllene oxide (1.6 and 2.2%),

camphene (1.6 and 1.3%) and limonene (1.2 and 1.1%) were the major compounds. Thus, the monoterpenes and sesquiterpenes were the predominant portions of the oils. Essential oils Keywords: Orthosiphon stamineus Essential oil composition -Caryophyllene -Humulene -Elemene 1-Octen-3-ol Caryophyllene oxide MIC Anti-fungal activity and methanol extract of O. stamineus and the derived fractions of hexane, chloroform, and ethyl acetate were tested for anti-fungal activity, which was determined by disc diffusion and minimum inhibitory concentration (MIC) determination methods. The oils, methanol extract and derived fractions of methanol extract displayed great potential of anti-fungal activity as a mycelial growth inhibitor against the tested phytopathogenic fungi such as Botrytis cinerea, Rhizoctonia solani, Fusarium solani, Colletotricum capsici and Phytophthora capsici, in the range of 49.370.3% and minimum inhibitory concentration ranging from 500 to 1000 g/ml. 2007 Elsevier B.V. All rights reserved.

1.

Introduction

Fungi have long been recognized as causal agents of plant diseases. Rice sheath blight (Rhizoctonia solani), grey mold rot (Botrytis cinerea), fruit rot (Fusarium solani), vascular wilt (F. oxysporum), water soaked spot (Sclerotinia sclerotiorum) and fruit rot (Phytophthora capsici) are important plant diseases (Saini and Sharma, 1978; Purdy, 1979; Lee and Rush, 1983; Agrios, 1988; Mullins et al., 1992; Rojo et al., 2007). Chemical fungicides are

known to be highly effective to control the postharvest diseases in various vegetables and fruits. However, they are not considered as long-term solutions due to the concerns associated with exposure risks, health and environmental hazards, residue persistence, and development of tolerance (Lingk, 1991; Radja Commare et al., 2002). The increasing recognition and importance of fungal infections and the difculties encountered in their treatment have stimulated the search for synthetic chemical fungicide alternatives. Essential oils

Corresponding author. Tel.: +88 2 8628913; fax: +88 2 8617946. E-mail address: dramzadh@gmail.com (M.A. Hossain). 0926-6690/$ see front matter 2007 Elsevier B.V. All rights reserved. doi:10.1016/j.indcrop.2007.11.008

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are made up of many different volatile compounds and have been shown to possess antimicrobial and fungicidal properties (Karmen et al., 2003; Ahmet et al., 2005). Essential oils and plant extracts are gaining increasing interest because of their relatively safe status, their wide acceptance by consumers, and their exploitation for potential multi-purpose functional uses (Sawamura, 2000; Ormancey et al., 2001). So, essential oils and plant extracts are one of the most promising groups of natural compounds for the development of safer anti-fungal agents. Orthosiphon stamineus Benth is used as a medicinal plant because of its diuretic, anti-fungal and bacteriostatic properties of leaves (Olah et al., 2003). Most of the papers dealing with this subject refer these effects to the content of potassium, inositol and lipophilic avones in Orthosiphon leaves (Schneider and Tan, 1973; Schut and Zwaving, 1993). In addition to the mentioned components, saponins, sterols, polyphenols, rosmarinic acid and ursolic acid and essential oil have been also detected (Stecher, 1976; Tezuka et al., 2000; Hossain et al., 2006). The essential oil, in some cases, is the reason for diuretic effects of plant drugs, has not yet been described in detail. Based on preliminary analyses, Awale et al. (2003) have reported that it mainly consists of oxygenated sesquiterpenes. However, there is no report available in the literature on the detailed analyses of essential oil of O. stamineus and its anti-fungal property. Therefore, the aim of the present study is (a) to examine the chemical composition of the essential oils isolated from the leaves and stems of O. stamineus by GCMS/MS; (b) to evaluate the anti-fungal activity of essential oils and methanolic extract of O. stamineus and its derived fractions of hexane, chloroform and ethyl acetate against certain important phytopathogens causing severe diseases in the plants.

temperature and the solvents from the combined extracts were evaporated by a vacuum rotary evaporator (EYELA N1000, Japan). The methanol extract was (5.3 g) suspended in water and extracted successively with hexane, chloroform and ethyl acetate to give hexane (1.97 g), chloroform (0.93 g) and ethyl acetate (0.78 g) and residual methanol fractions (0.58 g), respectively. Solvents (analytical grade) for extraction were obtained from commercial sources.

2.4.

GCMS/MS analysis

The GCMS/MS analysis of the essential oils was performed using a Waters GCMS/MS (Model 800) equipped with a ZB-1 MS-fused silica capillary column (30 m 0.25m i.d., lm thickness 0.25 m). For GCMS/MS detection, an electron ionization system with ionization energy of 70 eV was used. Helium gas was used as a carrier gas at a constant ow rate of 1 ml/min. Injector and mass transfer line temperature were set at 220 and 290 C, respectively. The oven temperature was programmed from 50 to 150 C at 3 C/min, then held isothermal for 10 min and nally raised to 250 C at 10 C/min. Diluted samples (1/100 (v/v), in methanol) of 1 l was manually injected in the splitless mode. The relative percentage of the oil constituents was expressed as percentage by peak area normalization. The identity of the components of the essential oils was assigned by comparison of their retention indices, relative to a series n-alkane indices on the ZB-1 capillary column and GCMS spectra from the Wiley 6.0 MS data and literature data and whenever possible, by co-injection with authentic compounds (Joulain and Konig, 1998; Adam, 2001).

2.5.

Microorganisms

2.
2.1.

Materials and methods

Plant material

The leaves and stems of O. stamineus were collected from the hilly area at Penang in Malaysia, in July 2003 and initially identied by morphological features and data base present in the library, School of Biology, University Sains Malaysia, Malaysia and a voucher specimen has been deposited at the Forest Research Institute, Malaysia (FRIM) with voucher number ZAS 1113.

The fungal cultures were obtained from the Herbal Laboratory, University Sains Malaysia, Malaysia (HL). Cultures of each fungal species were maintained on potato-dextrose-agar (PDA) slants and stored at less than 4 C. The fungal species used in the experiment were R. solani HL 325, B. cinerea HL 206, F. solani HL 115, Colletotricum capsici HL 410 and P. capsici HL 97.

2.6.

Preparation of spore suspension and test samples

2.2.

Isolation of the essential oils

The air-dried leaves and stems (250 g for each) of O. stamineus were subjected to hydrodistillation for 3 h using a Clevenger type apparatus. The oils were dried over anhydrous sodium sulphate and preserved in a sealed vial at 4 C until further analysis.

The spore suspension of B. cinerea, R. solani, F. solani, P. capsici and C. capsici were obtained from their respective 10daycold cultures, mixed with sterile distilled water to obtain a homogenous spore suspension of 1 107 spore/ml. Essential oils, methanol extract of O. stamineus and its derived fractions of hexane, ethyl acetate and chloroform were dissolved in methanol separately to prepare the stock solution with their respective known weights, which were further diluted to prepare test samples.

2.3.

Preparation of crude extracts

2.6.1. Determination of anti-fungal activity of essential oils and crude extracts

Petri dishes (9 cm diameter) containing 20 ml of PDA medium were used for anti-fungal activity assay, performed in solid media by disc diffusion method (Duru et al., 2003). Sterile Whatman paper discs of 6 mm diameter were pierced in the

The air-dried leaves and stems of O. stamineus were pulverized into powdered form. The dried powder (50 g) was extracted three times with 70% methanol (3200 ml) at room

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agar plates, equidistant and near the border, where the essential oils 5 l (1000 ppm) and methanol extract and its derived fraction samples 7.5 l (1500 ppm) were used separately. A disc of fungal inoculum 6 mm in diameter was removed from a pre-grown cultures of all the fungal strains tested and placed upside down in the centre of the Petri dishes. The plates were incubated at less than 30 C for 7 days, time by which the growth of control would have reached the edges of the plates. Growth inhibition of each of the fungal strains was calculated as the percentage of inhibition of radial growth relative to the control along with anti-fungal effect on fungal mycelium. The plates were used in triplicates for each treatment. Growth inhibition of treatment against control was calculated by percentage, using the following formula: Inhibition ratio (%) = 1 mycelium growth of treatment (mm) 100 mycelium growth of control (mm)

Monoterpene and sesquiterpene hydrocarbons were the characteristic constituents of the oils of O. stamineus. -Pinene, 1,8-cineol, borneol, linalool, camphor, eugenol, p-cymene, carvone, bornyl acetate and -cadinene were also found to be the minor components of O. stamineus leaves and stems oils in the present study.

3.2. Anti-fungal activity of essential oils and crude extracts

The leaves and stems oils of O. stamineus exhibited a moderate to high anti-fungal activity against all the tested fungi except P. capsici. No inhibition effect was observed against P. capsici. At the concentration of 5 l (1000 ppm), the essential oils showed potent inhibitory effect on the growth of B. cinerea (64.066.0%), R. solani (56.364.0%), F. solani (63.3%) and C. capsici (65.368.0%), as shown in Table 2. Also, the crude methanol extract and its derived hexane, chloroform and ethyl acetate fractions showed mycelium growth inhibition against some of the phytopathogens but not for all. According to the results reported in Table 3, methanol extract of O. stamineus showed higher anti-fungal effect than essential oils against R. solani (70.3%), F. solani (68.0%), and C. capsici (70.0%), respectively. The crude methanol extract and its hexane, ethyl acetate and chloroform fractions did not show any inhibitory effect against P. capsici. As can be seen from Table 3, derived fractions of methanol extract showed moderate anti-fungal activity against some of the fungi tested. Hexane fraction showed maximum anti-fungal activity against R. solani and C. capsici (58.363.3%), but no inhibition was observed against F. solani and P. capsici, whereas, chloroform fraction showed relatively better anti-fungal effect against R. solani and F. solani (65.568.3%), as compared to ethyl acetate fraction.

2.6.2.

Minimum inhibitory concentration (MIC)

The minimum inhibitory concentration of essential oils and crude methanol extract and its derived fractions was determined by two-fold dilution method against B. cinerea, R. solani, F. solani, P. capsici and C. capsici (Murray et al., 1995). Samples were dissolved in methanol according to their respective known weights. The solutions were serially diluted with methanol and were added to PDA to nal concentrations of 125, 250, 500, and 1000 g/ml, respectively. A 10- l spore suspension of each test strain was inoculated in the test tubes in PDB medium and incubated for 57 days at less than 30 C. The control tubes containing PDA medium were inoculated only with fungal suspension. The minimum concentration at which no visible growth was observed was dened as the MIC, which was expressed in g/ml.

2.7.

Statistical analysis 3.3. Minimum inhibitory concentration

The essential oils, methanol extract and derived fractions of methanol extract were assayed for anti-fungal activity. Each experiment was run in triplicate, and mean values were calculated. A t-test was computed for the statistical signicance of the results.

3.
3.1.

Results
Chemical composition of essential oils

GCMS analyses of the oils led to the identication of 69 different compounds, representing 97.6 and 97.4% of the total oils from leaves and stems, respectively. The identied compounds are listed in Table 1 according to their elution order on a ZB-1 capillary column. The oils contain a complex mixture consisting of mainly oxygenated monoterpene and sesquiterpene hydrocarbons. The major compounds detected in the leaves and stems oils, respectively were -caryophyllene (24.0 and 35.1%), -humulene (14.2 and 18.4%), -elemene (11.1 and 8.5%), 1-octen-3-ol (8.2 and 7.0%), -bourbonene (3.4 and 3.0%), -pinene (2.1 and 1.7%), caryophyllene oxide (1.6 and 2.2%), camphene (1.6 and 1.3%) and limonene (1.2 and 1.1%) (Table 1).

According to the results given in Table 2, MIC of essential oils was found to be more effective against B. cinerea and F. solani (500 g/ml for each) as compared to that of R. solani and C. capsici (1000 g/ml for each). On the other hand, the crude methanol extract of O. stamineus and its chloroform fraction were found more susceptible than hexane and ethyl acetate fractions against the tested fungi (Table 4). As control, methanol did not affect the growth of sample strains at the concentration used in this study. The sub-inhibitory concentrations dened as the lowest concentrations of methanol extract against R. solani (1000 g/ml), C. capsici (1000 g/ml), B. cinerea (500 g/ml) and F. solani (500 g/ml) but no inhibition was observed against P. capsici, whereas chloroform fraction resulted in low inhibition of visible growth against B. cinerea, R. solani and C. capsici (1000 g/ml for each). Chloroform fractions did not show any inhibition against P. capsici. Ethyl acetate fraction showed the inhibition against B. cinerea and F. solani with their respective MIC values of 500 and 1000 g/ml, whereas hexane fraction did not show desirable results against all the phytopathogens tested.

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Table 1 Percentage composition of the volatile leaves and stems oils of Orthosiphon stamineus RIa
780 826 843 856 884 885 927 932 938 961 963 966 972 980 1003 1005 1009 1017 1028 1044 1054 1061 1074 1076 1085 1100 1105 1108 1129 1134 1136 1137 1148 1178 1179 1182 1196 1200 1225 1228 1230 1274 1278 1280 1289 1292 1300 1312 1322 1332 1345 1366 1375 1379 1380 1388 1392 1398 1414 1431 1447 1448 1492 1494 1505 1546 1556 1561 1816 Total
a

Compound
Hexanal trans-2-Hexanal cis-3-Hexen-l-ol Hexan-1-ol 4-Heptenal Heptenal Benzaldehyde -Pinene Camphene l-Octen-3-ol -Pinene 3-Octanol 2-Pentenyl furane 2-Amylfurane p-Cymene 1,8-Cineol Limonene Acetophenone cis-2-Octenal Phenylacetaldehyde trans,cis-Octa-3,5-dien-2-one cis-Linalooloxide trans,trans-Octa-3,5-dien-2-one Linalool trans-Linalooloxide Undecan 2,6,6-Trimethyl-2-cyclohexe-l,4-dione Perillen Camphor -Terpineol trans -2-(cis)-6-Nonadienale Menthone Isomenthone Methylchavicol Borneol Decanal Naphthalene Dodecane Cittonellol Carvone -Cyclocitral trans-Anethol Isobornylacetate Safranal 1-Methylnaphthalene Bornyl acetate Tridecan 2-Methylnaphthalene trans, trans-Deca-2,4-dienal -Elemene -Cubebene Damascenone -Copaene -Bourbonene Eugenol -Elemene Methyleugenol cis-Caryophyllene -Carryophyllene Geranylacetone -Humulene -Ionone Germacrene D -Muuiolene -Cadinene Germacrene B Dehydroionone Caryophyllene oxide Hexahydrofamesylacetone

Leaves (%)
1.07 0.98 0.01 0.05 0.78 0.97 0.99 0.72 1.6 8.2 2.1 1.42 1.11 0.56 0.67 0.82 1.2 0.23 1.48 1.65 0.92 0.13 0.60 0.44 0.39 0.12 0.03 0.09 0.65 0.23 0.33 0.55 0.77 0.72 0.51 0.88 1.00 0.49 0.52 0.55 0.19 0.22 0.03 0.08 0.63 0.12 0.71 0.11 0.03 0.88 0.27 0.18 0.52 3.4 0.47 11.1 0.31 0.03 24.0 0.23 14.2 0.11 0.21 0.61 0.37 0.03 0.16 1.6 0.08 97.6

Stems (%)
1.09 0.53 0.44 0.08 0.42 0.54 1.01 0.09 1.3 7.0 1.7 1.03 0.73 0.88 0.45 0.14 1.1 0.62 0.78 0.87 0.02 0.27 0.49 0.21 0.22 0.02 0.21 0.10 0.23 0.41 0.12 0.06 0.34 0.51 0.33 0.15 0.28 0.10 0.69 0.34 0.22 0.09 0.07 0.10 0.54 0.01 0.39 0.07 0.08 0.18 0.48 0.08 0.33 3.0 0.21 8.5 0.21 0.05 35.1 0.43 18.4 0.34 0.09 0.42 0.12 0.01 0.10 2.2 0.01 97.4

Retention index relative to n-alkanes on ZB-1 capillary column.

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Table 2 Anti-fungal activity of the leaves and stems essential oils 5 l (1000 ppm) of O. stamineus Fungal strains Leaves essential oil Mycelial growth inhibition a mm
B. cinerea HL 206 R solani HL 325 F. solani HL 115 P. capsici HL 97 C. capsici HL410 16.0 0.4 19.1 0.4 16.4 0.8 nd 16.0 0.7

Stems essential oil MIC ( g/ml) Mycelial growth inhibition a mm

500 1000 500 na 1000 16.0 0.4 16.5 0.6 16.5 0.4 nd 14.5 0.8

MIC ( g/ml)

(%)
64.0 1.2 56.3 0.6 63.3 1.1 nd 65.3 1.1

(%)
66.0 0.6 64.0 1.5 63.3 1.1 nd 68.0 1.7 500 1000 500 na 1000

nd: no detection of anti-fungal activity; na: not applicable.

a

Values are represented as the mean S.D. of three experiments.

Table 3 Anti-fungal activity of methanol extract and its derived fractions 1.5 l (1500 ppm) of O. stamineus Fungal strains CME mm
B. cinerea HL 206 R solani HL32-5 F.solani HL 115 P. capsici HL 97 C. capsici HL 410 16.0 0.5 13.5 0.6 14.5 0.3 nd 13.5 0.7

Mycelial growth inhibition a HAF (%)

66.0 1.1 70.3 1.5 68.0 1.0 nd 70.0 1.7

EAF (%)
49.3 0.6 63.3 1.1 nd nd 58.3 1.1

CHF (%)
64.0 1.1 nd 55.0 1.5 nd nd

mm
23.0 0.3 17.0 0.4 nd nd 19.0 0.5

mm
16.1 0.3 nd 20.3 0.6 nd nd

Mm
16.1 0.4 15.0 0.3 15.5 0.4 nd nd

(%)
63.5 1.2 68.3 0.4 65.5 0.4 nd nd

CME: crude methanol extract; HAF: hexane fraction; EAF: ethyl acetate fraction; CHF: chloroform fraction; nd: no detection of anti-fungal activity.
a

Values are represented as the mean S.D. of three experiments.

4.

Discussion

The increasing social and economic implications caused by fungi means there is a constant striving to produce safer food crops and to develop new anti-fungal agents. In general, plant-derived essential oils are considered as non-phytotoxic compounds and potentially effective against plant pathogenic fungi (Pandey et al., 1982). In recent years, interests have been generated in the development of safer anti-fungal agents such as plant-based essential oils and extracts to control phytopathogens in agriculture (Costa et al., 2000). Several publications have documented the anti-fungal activities of

Table 4 Minimum inhibitory concentrations ( g/ml) of methanol extract and its derived fractions of O.s stamineus against tested phytopathogens Fungal strains CME
B. cinerea HL 206 R. solani HL 325 F.solani HL 115 P. capsici HL 97 C. capsici HL 410 500 1000 500 nd 1000

MIC ( g/ml) HAF

nd nd nd nd nd

EAP
500 nd 1000 nd nd

CHF
1000 1000 500 nd 1000

CME: crude methanol extract; HAF: hexane fraction; EAF: ethyl acetate fraction; CHF: chloroform fraction; nd: no detection of antifungal activity.

essential oils and plant extracts (Morris et al., 1979; Yousef and Tawil, 1980; Bajpai et al., 2007). Thus essential oils are promising natural anti-fungal agents with potential applications in agro-industries to control plant pathogenic fungi causing severe destruction in crops. The hydrodistillation of the leaves and stems of O. stamineus gave dark yellowish oils with the major components of the oils having oxygenated monoterpenes and sesquiterpenes, and their respective hydrocarbons. In recent years, several researchers have reported that monoterpene and sesquiterpene hydrocarbons and their oxygenated derivatives are the major components of essential oils of plant origin, which have enormous potential to strongly inhibit microbial pathogens (Gudzic et al., 2002; Cakir et al., 2004). In general, the active antimicrobial compounds of essential oils are terpenes, which are phenolic in nature, it would seem reasonable that their antimicrobial or anti-fungal mode of action might be related to that of other compounds. The essential oils of O. stamineus showed remarkable antifungal effect against all the fungi tested except P. capsici. This activity could be attributed to presence of -caryophyllene (24.0 and 35.1%), -humulene (14.2 and 18.4%), -elemene (11.1 and 8.5%), 1-octen-3-ol (8.2 and 7.0%), -bourbonene (3.4 and 3.0%), -pinene (2.1 and 1.7%), caryophyllene oxide (1.6 and 2.2%), camphene (1.6 and 1.3%) and limonene (1.2 and 1.1%), which signicantly (65%) inhibited the growth of all the phytopathogens tested, and/or other major and minor oxygenated monoterpenes and sesquiterpenes present in the oils. Some earlier papers on the analysis and anti-fungal properties of the essential oils of some species of various genera

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have shown that they have a varying degree of growth inhibition effects against some Fusarium, Botrytis and Rhizoctonia species due to their different chemical composition (AlvarezCastellanos et al., 2001; Singh et al., 2002; Bouchra et al., 2003). Bouchra et al. (2003) reported that the oils of seven Moroccan Labiatae, which consist mainly carvacrol, linalyl acetate and tymol as major components, exhibited a complete mycelial inhibition effect on the growth of B. cinerea . On the other hand, the oils of Pistacia versa, P. terebinthus and P. lentiscus had moderate activities at 750 ppm doses against R. solani (AlvarezCastellanos et al., 2001). In spite of this, most of these oils are available for purchase as whole or as a part of pharmaceutical or cosmetic products, indicating that toxic properties do not prohibit their use. However, the ongoing investigation of toxic or irritant properties is imperative, especially when considering any new products for human use, be them medicinal or otherwise. In this study, the essential oils and the different extracts showed varying anti-fungal activity against various plant pathogenic fungi, which could be attributed to the presence of phenolic compounds and oxygenated monoterpenes and sesquiterpene hydrocarbons (Guillen and Manzanos, 1998; Shunying et al., 2005). In our opinion, major components of O. stamineus essential oils, -caryophyllene, caryophyllene oxide, -humulene, -pinene, limonene, -elemene, and 1-octen-3ol have key roles for their anti-fungal activities. Anti-fungal activities of these compounds have been reported by others (Costa et al., 2000; Hammer et al., 2003; Filipowicz et al., 2003; Okull et al., 2003; Sacchetti et al., 2004; Deba et al., 2008). On the other hand, the components in lower amount such as -pinene, 1,8-cineol, linalool, camphor, eugenol, p-cymene, carvone, bornylacetate and cadinene also contributed to antifungal activity of the oils (Sokovic et al., 2002; Filipowicz et al., 2003; Hammer et al., 2003; Sacchetti et al., 2004; Deba et al., 2008). It is also possible that the minor components might be involved in some type of synergism with the other active compounds (Marino et al., 2001). Therefore, it would also be interesting to study the effects of essential oils and crude extracts of O. stamineus against other important fungi for developing new anti-fungal agents to control serious fungal diseases in plant, animal and human beings. Thus, O. stamineus could become an alternative to synthetic fungicides for using in agro-industries and also to screen and develop such novel types of selective and natural fungicides in the treatment of many microbial phytopathogens causing severe destruction to crop, vegetable and ornamental plants.

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