international journal of pharma and bio sciences issn 0975 ... · pdf fileworld after soybean...

Int J Pharm Bio Sci 2014 Oct; 5(4): (B) 1010 - 1019

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Research Article Pathology

International Journal of Pharma and Bio Sciences ISSN

0975-6299

ANTI-MILDEW ACTIVITY OF METHANOLIC EXTRACT FROM SELECTED

MEDICINAL PLANTS AGAINST PLASMOPARA HALSTEDII (FARL.) BERL. & DE

TONI INCITANT OF SUNFLOWER DOWNY MILDEW DISEASE

R. GIRIJAMBA1, P. HARIPRASAD2, S. BRIJESH SINGH1 AND S. R. NIRANJANA1*

1

Department of Biotechnology, University of Mysore, Manasagangotri, Mysore-570 006, Karnataka, India. 2

Centre for Rural Development and Technology Indian Institute of Technology Delhi Hauz Khas, New Delhi 110016

ABSTRACT

Methanolic extracts of eight medicinal plants were screened for their anti-mildew activity against Plasmopara halstedii, incitant of downy mildew of sunflower. Extracts of Morinda citrifolia, Zingiber officinale, and Tinospora cordifollia were found to be best in reducing the sporulation of P. halstedii in both the whole leaf and leaf disc method. Further; the phytochemical analysis of these extracts revealed the presence of alkaloids, glycosides, steroids, flavonoids, reducing sugars, phenolics, tannins, saponins and terpenoids. Different compounds from the plant extracts were partially purified through preparative thin layer chromatography and analyzed for their anti-mildew activity following leaf disc method. The assay confirmed that bioactive compound from M. citrifolia (Rf, 0.47), Z. officinale (Rf, 0.6) and T. cordifollia (Rf, 0.12) were antimildew in nature. In vitro sporangiosporicidal assay revealed that, partially purified antimildew compounds significantly reduced the viable sporangiospores and also inhibited the release of sporangiospores from sporangia. KEY WORDS: Sunflower, downy mildew, Plasmopara halstedii, anti-mildew activity.

*Corresponding author

S. R. NIRANJANA

Department of Biotechnology, University of Mysore,

Manasagangotri, Mysore-570 006, Karnataka, India.



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INTRODUCTION The Sunflower (Helianthus annuus L.) is one of the major edible oil seed crops grown across world after soybean and groundnut. It is cultivated over an area of 2.4 million hectares with a 1.44 million tones of production and 608 kg/ha of productivity1. Sunflower oil is rich source of linolic and oleic acid2, used to treat wounds in animals3. Edible oils from sunflower extracted seeds are important in foods which provide characteristic taste, odor and texture for processed food. Also, high oleic sunflower oil is considered as excellent alternative for existing edible oils to manage heart diseases. It is being used for cosmetics, pharmaceutical, lubricants, gasoline blend and other purposes. Many fungal, bacterial and viral diseases are reported as major impediment in Sunflower production. Among them, downy mildew of sunflower caused by Plasmopara halstedii (Farl.) Berl. and de Toni, which has been recognized as potentially destructive disease in major sunflower producing countries due to its systemic nature of infection. In India, downy mildew of sunflower was first reported from Marathwada region of Maharashtra around 1986 with yield losses up to 80%4. The downy mildew disease of sunflower has been dispersed mainly by seed to all sunflower-growing countries5-6. The infected seedlings become dead, but those which are survived have produce stunted plants with erect, platform heads and no flowers7. Leaves of the affected plants grow abnormally thick, curled, downward and mottled. Downy growth of the fungus consisting of sporangiophores and sporangia are observed on the lower side of the leaves whereas the corresponding upper surface of the leaves appear chlorotic yellow. Further sporangia give rise to biflagellate zoospores which are motile and cause secondary infection. The disease is extremely difficult or impossible to eradicate once it is established in an area8. Realizing the importance of the downy mildew disease in sunflower, priority has been given to manage the disease by using chemicals (metalaxyl as seed treatment and as foliar spray or soil treatment). However, chemicals have limited success and cause undesirable problems. Large scale utilization of chemicals has resulted in deterioration of soil fertility, residual toxicity, development of resistance by plant

pathogen, loss in biodiversity, increasing cost of plant protection and an increase in human health hazard by way of environmental pollution. Therefore, to alleviate all these ill effects, environmentally safe and long lasting eco-friendly disease management strategies are needed. Use of resistant varieties, crop rotation and eradicating alternative hosts are found useful in managing this disease. But, these methods had their own limitations. On the other hand use of phytochemicals with antimicrobial activity has been considered as safe and ecofriendly method for management of agriculturally important diseases9. Evaluation and utilization of crude/purified bioactive compounds from medicinal plants in agriculture against phytopathogens has been frequently reported. But, use of phytochemicals for the management of oomycetes diseases was less explored. Hence, the present study was aimed to develop an ecofriendly strategy for the management of sunflower downy mildew diseases by evaluating the bioactive compounds from selected medicinal plants for their anti-mildew and sporangiosporicidal activity.

MATERIALS AND METHODS Plant Materials Sunflower seeds (Cv. Morden) susceptible to downy mildew disease was collected from National Seed Corporation, Mysore, Karnataka. The seed samples were surface sterilized with 0.2 % sodium hypochlorite for 2 min and subsequently washed with distilled water thrice. The surface sterilized seeds were used throughout the experiments. Medicinal plants like Centella asiatica (L.) Urban, Clerodendrum inerme (L.) Gaertn, Eucalyptus globules Labill., Morinda citrifolia L., Tinospora cordifolia (Thunb.) Miers, Phyllanthus amarus Schum and Thonn., Zingiber officinale Roscoe and Ocimum sanctum L., commonly grown in and around Mysore district were collected during June to December, 2012. The plant species were identified taxonomically at the Department of Botany, University of Mysore (Table 1).



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Preparation of plant extracts The collected plant materials were shade-dried and ground to a coarse powder using a mechanical blender. Crude compound were extracted using methanol in Soxhlet apparatus. The crude extracts obtained were dried to powder. Different concentration (1, 2, 3, 5, 7 and 10 %) was prepared by dissolving it in minimum quantity of DMSO and make up to desired volume with sterile distilled water. Test organisms A sick plot of downy mildew pathogen Plasmopara halstedii has been maintained in the Department of Biotechnology, University of Mysore, Mysore, India, since the last 10 years (N 24º18’, E 79º 26’, 903 m altitude). In order to harvest inoculum for the experiment, susceptible Cv. Morden was grown in sick plot for 30 days. For whole leaf and leaf disc assay, infected leaves showing profuse growth of sporangiophore and sporangia were collected in late evening. Determination of anti-mildew activity of medicinal plant extracts Anti-mildew activity of the medicinal plant extracts against P. halstedii was investigated by leaf disc and whole leaf method10-12. Leaves with disease symptoms were collected from infected plants grown in sick plot and washed in distilled water. The excess water was then removed by blot drying. In leaf disc method, leaves were cut in to small discs of 10 mm diameter by using sterilized cork borers and subsequently immersed in plant extracts of different concentrations for 5 min. The treated discs were then incubated for 12-14 h in moist chambers by facing abaxial surface upside at 22±1ºC in the dark. In the whole leaf method, downy mildew infected leaves were washed to remove the existing sporangia and surface wetness was removed by blot drying. Different concentrations of the plant extract was applied on the abaxial surfaces of the leaf by coating with sterilized brush. After incubating in a moist chamber for 12-14 h, the leaf discs and whole leaves were analyzed for sporulation under stereobinocular microscope and categorized as explained below. The anti-mildew activity was evaluated on following scale: Complete inhibition (+++), Moderate inhibition (++), least inhibition (+) and no inhibition (-).

Phytochemical analysis of the selected positive medicinal plant extracts The Phytochemical analysis of methanolic extracts of M. citrifolia, T. cordifolia and Z. officinale was carried out following the standard procedures as follows, Alkaloid test: 2 % HCl was added to 0.5 ml of crude extract followed by steaming for 5 min. Further, to the filtrate, 6 drops of Mayer’s reagent was added and observed for creamish or brownish red precipitate which indicates the presence of alkaloid13. Tannins Test: 0.5 ml of extract was mixed with 1 ml of distilled water and heated on water bath followed by the addition of 5 % ferric chloride solution. Appearance of a dark green colouration indicates the presence of tannins14. Terpenoid and Steroid test: 0.5 ml of extract was treated with 0.5 ml of acetic anhydride and 0.5 ml of chloroform followed by the slow addition of (100 µl) concentrated solution of sulphuric acid. Appearance of red violet color indicates the presence of terpenoid and green bluish color indicates the presence of steroids15. Flavonoid Test: 1 g of metal magnesium was

added to a 0.5 ml of warm crude extract followed by the addition of 5-6 drops of concentrated hydrochloric acid. The development of red color indicates the presence of flavonoids whereas orange color indicates flavones15. Glycosides test: 0.5 ml of methanolic extract was mixed with 1 ml of glacial acetic acid, few drops of ferric chloride and concentrated sulphuric acid. Development of reddish brown color in the upper layer indicates the presence of glycosides15. Reducing sugar test: 0.5 ml of extract was mixed with 1 ml of water followed by addition of 5-8 drops of Fehling’s solution and then the mixture is heated slightly. Development of brick red precipitate color indicates the presence of reducing sugar13. Saponin test: 0.5 ml of the extract taken in separate test tubes were vigorously shaken for 2 mins and boiled in a water bath.



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Development of froth indicates the presence of saponins in the test sample13. Phenolics test: To 0.5 ml of the extract, a few drops of 5 % (v/v) Glacial acetic acid and 5 % sodium nitrate solutions were mixed. Development of muddy yellow/olive brown/niger brown/deep chocolate colour indicates the presence of phenolics compounds in test solution14. Thin layer chromatography analysis of methanolic extracts of the selected medicinal plant The crude plant extracts which showed the anti-mildew activity were further separated on precoated 20 x 20 cm TLC Silica gel 60 F 254 (Merck). Ten µl of each extract was spotted on TLC plate at equal distance and the chromatogram was developed with standardization solvent system which is as follows, M.citrifolia (chloroform : toluene: methanol, 9.5:0.3:0.2), T. cordifolia (toluene : methanol,9.25:0.75) and Z.officinale (chloroform : benzene, 9.5:0.5).The developed chromatogram was observed under visible and Ultra violet light, and documented. Preparative TLC was conducted as explained above except the silica gel plates used were of 2 mm thickness. The spots at different Rf value were scraped off and eluted using methanol. The eluted compound was tested for their antimildew activity by leaf disc assay at 1% concentration. Sporangiosporicidal Assay Leaves of sunflower showing profuse sporulation on the abaxial surface were collected from the plants maintained at the sick plot in the evening hours and washed thoroughly under running tap water to get rid of the previous crop of sporangia. The leaves were then blot-dried and kept in a moist chamber overnight for sporulation. The next morning, the newly formed sporangia were harvested in distilled water and the sporangial concentration was adjusted to 4×104

zoospores/ml16 using haemocytometer and used as inoculum. 100 µl of sporangial suspension of P. halstedii and 100 µl of different concentrations of extracts were added (final concentration; 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 25 and 50 µg/ml) and incubated along with 20 µl of Triphenyltetrazolium chloride

(TTC) solution in 0.5 ml eppendorf tubes for 12 h in a humid chamber. Following incubation, the mixture was centrifuged at 8000 rpm for 8 mins and the pellet was washed thrice with sterile distilled water. The pellet was then dissolved in 300 µl of sterile distilled water. The number of sporangia without zoospore (viable), colored sporangia with zoospores (viable) and uncolored sporagia with zoospores (non viable) were counted using hemocytometer. To quantify the formazane formed in living cells, the pellet was mixed with 300 µl of 95 % ethanol and incubated in a waterbath at 85°C for 30 min. The mixture was further centrifuged at 8000 rpm for 8 min. 100 µl supernatant was transferred to a microtiter plate and read at 485 nm against respective controls. Sporangiosporicidal assay was performed in triplicates for each plant extract and the average was tabulated 17. Sporangia and zoospores incubated in distilled water served as control.

RESULTS Anti-mildew activity of medicinal plants against sunflower downy mildew Among methanolic extracts of eight medicinal plants, M. citrifolia, T. cordifolia and Z. officinale were found best in inhibiting the sporulation of P. halstedii in both the leaf disc and whole leaf method. In all positive extracts, as the concentration increases the anti-mildew activity was also found increasing up to 5% above which there was no significant (p≤0.05) difference between concentrations (Table 1). Phytochemical analysis The phytochemical analysis of the methanolic extracts of all the three selected medicinal plants revealed the presence of alkaloids, glycosides, steroids, flavonoids, reducing sugars and phenolics in M. citrifolia; alkaloids, tannins, steroids, phenolics, glycosides, flavonoids and saponins in T. cordifolia; and tannins, alkaloids, glycosides, terpenoids, flavonoids, saponins and phenolic in Z. officinale (Table 2).

Anti-mildew activity of partially purified compounds TLC analysis of methanolic extracts of M. citrifolia, T. cordifolia and Z. officinale revealed the presence of compounds with varied Rf



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values. Among them the compounds with Rf values 0.47 (M. citrifolia), 0.6 (Z. officinale) and 0.12 (T. cordifollia) were significantly inhibited the sporulation in leaf disc method in comparison with other compounds and the controls (Fig 1, Table 3). Sporangiosporicidal assay The effect of partially purified bioactive compounds on the viability of sporangia of P. halstedii was determined following TTC assay. The percentage of viable sporangia decreases

as the concentrations of anti-mildew compounds increases as compared with that of respective controls. Concurrently, percentage non-viable sporangia increase. The result of the TTC assay was tabulated in Fig. 2, 3 and 4. Among the three extracts tested, anti-mildew compound from M. citrifolia significantly inhibited the release of sporagiospores from sporangia and also reduced the viable sporangia (IC50, 6.25) in comparison with T. cordifolia (IC50, 8.16) and Z. officinale (IC50, 8.32).

Table 1

Antimildew activity of methanolic extract of selected medicinal plants.

Plant species Family Part used

Disc method Whole leaf method

Concentration (%)

1 2 3 5 7 10 1 2 3 5 7 10

Centella asiatica Apiaceae Leaf - - - - - + - - - - - -

Morinda citrifolia Rubiaceae Bark + + ++ +++ +++ +++ - + ++ +++ +++ +++

Cleodendrum inermae Verbinaceae Leaf - - - - - + - - - - - -

Ocimum sanctum Lamiaceae Leaf - - - - - - - - - - -

Zingiber officinale Zingiberaceae Rhizome - + ++ +++ +++ +++ - + ++ +++ +++ +++

Tinospora cordifolia Menispermaceae Stem - + ++ +++ +++ +++ - + + +++ +++ +++

Eucalyptus globosus Myrtaqceae Leaves - - - - - + - - - - - +

Phyllanthus niruri Euphorbiaceae Leaf - - - - - - - - - - - -

Antimildew activity was evaluated by following scale, Complete inhibition (+++), Moderate inhibition (++), least inhitibion (+) and no inhibition (-).

Table 2

Phytochemical analysis of methanolic extracts with anti-mildew activity

Phytochemical tests Morinda citrifolia Tinospora cordifolia Zingiber officinale

Tannin - + +

Alkaloid + + +

Glycoside + + +

Steroid + + -

Terpenoid - - +

Phenolics + + +

Flavonoids + + +

Reducing sugar + - -

Saponins - + + + = Positive for test; - = Negative for test.

Table 3

Anti-mildew activity of partially purified fractions of selected medicinal plants

Fractions a b c d E F G

Morinda citrifolia

RF value 0.06 0.28 0.38 0.47 0.77 0.96 0.97

Inhibition - + ++ +++ + + -

Tinospora cordifolia

RF value 0.04 0.12 0.23 0.63 0.76 0.83 -

Inhibition - +++ ++ ++ + + -

Zingiber officinale

RF value 0.6 0.12 0.75 0.83 - - -

Inhibition + +++ ++ - - - - Anti-mildew activity was evaluated by following scale, Complete inhibition (+++), Moderate inhibition (++), least inhibition (+) and no inhibition (-).



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Figure 1

Thin layer chromatography analysis of crude methanolic extracts of selected medicinal plants and leaf disc bio-assay for anti-mildew activity of bands with different Rf value. a. TLC chromatogram of Tinospora cordifolia and fraction b with Rf value 0.12 showing inhibition of sporangiophore formation (inner figure, control leaf disc with sporangiophore), b. TLC chromatogram of Morinda citrifolia bark and fraction d with Rf value 0.47 showing inhibition of sporangiophore formation and c. TLC chromatogram of Zingiber officinale rhizome and fraction b with Rf value 0.6 showing inhibition of sporangiophore formation.



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Figure 2 Sporangiosporicidal activity of partially purified compound from M. citrifolia.

Note: Viable cells = Colored sporangia + empty sporangia.

Figure 3

Sporangiosporicidal activity of partially purified compound from M. citrifolia.


Figure 4

Sporangiosporicidal activity of partially purified compound from Z. officinale.




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DISCUSSION In the present study methanolic extracts of eight medicinal plants were screened for their anti-mildew activity against sunflower downy mildew disease. Plant extracts with antimicrobial activity were used for the management of various plant diseases, and new plants and their extracts are frequently reported for the above mentioned activities18-20. But the management of oomycetes phytopathogen is a challenge as it behavious differently from other fungal pathogen. Considering use of plant extract as ecofriendly approach to manage oomycetes disease, it is important to screen antimildew activity of such extracts. The methanolic extract of forty plant species was evaluated for their antisporulating activity against Sclerospora graminicola, the causative agent of pearl millet downy mildew21

. Among them the extracts of 11 plant species such as Agave Americana, Artemisia pallens, Citrus sinensis, Dalbergia latifolia, Helianthus annus, Murraya koenigii, Ocimum basilicum, Parthenium hysterophorus, Tagetes erecta, Thuja occidentalis and Zingiber officinale exhibited the antisporulating activity. Further, 40 more aqueous extracts of plants were screened for their antimildew acitivty12. According to their results, extracts of Allium sativum, Clematis gouriana, Evolvulu salsinoides, Mimus opselengi, Parthenium hysterophorus, Piper nigrum and Tagetes erecta showed significant antisporulating activity. Similarly in our studies among the eight methanolic extracts, three plant species viz., M. citrifolia, Z. officinale and T. cordifolia inhibited the sporulation of P. halstedii in both the leaf disc and the whole leaf method. The anti-mildew activity of the selected medicinal plants due to presence of one or many bioactive compound and their antimicrobial activity were reported earlier. Plants belonging to family Zingiberaceae are known to contain many essential oils, including terpenes, alcohols, ketones, flavonoids and phytoesterogens having antimicrobial activity 22-24. A number of major components have been identified in the M. citrifolia, which includes scopoletin, octoanoic acid, terpenoids, alkaloids, anthraquinones, β-sitosterol, carotene, flavone glycosides, linoleic acid, alizarin, acubin, L-asperuloside, caproic acid, caprylic acid, ursolic acid, rutin, and

peroxeronine25-28. Important group of medicinal plants belonging to Tinospora species are known to contain protoberberine alkaloids, diterpenoids, flavonoids, steroids, certain lignans and polysaccharide29-30. The protoberberine alkaloids namely, berberine and jatrorrhizine are of considerable value and have been reported to possess antimicrobial activity31. In our studies, phytochemical analysis of extracts with anti-mildew activity revealed the presence of alkaloids, glycosides, tannins, terpenoids, steroids, flavonoids, reducing sugars and phenolics and saponins (Table 2). Further analysis of partially purified compound from preparative TLC for their anti-mildew activity revealed the presence of more than one compound with potential to suppress the sporulation. Bioactive compounds with high antimildew activity were further evaluated for their sporangiosporicidal activity. From sporangiosporicidal assay it was found that, antimildew compound from M. citrifolia showed significantly higher activity with IC50 value of 6.25 followed by T. cordifolia and Z. officinale and is evident by reduced colored sporangia (viable) and empty sporangia (viable) and increased non-colored sporangia (non-viable).

CONCLUSION From the results obtained it could be concluded that, methanolic extracts of M. citrifolia, T. cordifolia and Z. officinale were better sources for the development of ecofriendly management strategy against downy mildew disease of sunflower. Significantly higher sporangisporicidal activity of M. citrifolia is an added advantage to prevent secondary spread of disease under field conditions. Further studies regarding the identity of bioactive compound, stability, preparation of formulations and field studies should be conducted in order to standardize techniques for downy mildew disease management in sunflower.

ACKNOWLEDGEMENT The authors are thankful to the University Grants Commission, New Delhi, for the financial assistance and Government of India, New Delhi. Chairman, Department of Biotechnology, University of Mysore for kind support.



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international journal of pharma and bio sciences issn 0975 ... · pdf fileworld after soybean...

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