Chapter 3 Screening of medicinal plant extracts against Plasmopara halstedii causing downy mildew disease of sunflower 3.

Transcription

1 Chapter 3 Screening of medicinal plant extracts against Plasmopara halstedii causing downy mildew disease of sunflower 3.1: Introduction India is a vast country having major botanicals, abundant flora and fauna. About 80% of the world s population still depends on the ayurvedic system of medicines because of the adverse effects of the conventional medicines (WHO). According to Indian Vedas the use of herbal products and aqueous extract of different plant parts are used for healing many infectious diseases. A large number of plants and their products are the source of anti-cancer agents. ((Loi, et al., 2005). Plants extracts are used as a source of medicine due to its antimicrobial activity. (Shrivastava et al., 1996). Now days the loss of crop yield in the world is due to pathogenic diseases caused by the microorganisms. (Savary et al., 2006; Montesinos, 2007). On the other hand use of plant extracts with antimicrobial activity has been considered as safe and ecofriendly method for management of agriculturally important diseases (Cowan, 1999). Evaluation and utilization of crude or purified bioactive compounds from medicinal plants are frequently reported. But the use of plant extracts 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 activity. 3.2: Materials and methods 3.2.1: Plant Materials Sunflower seeds (cv. Morden) susceptible to downy mildew disease were 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 35

2 (Thunb.) Miers, Phyllanthus amarus Schum and Thonn., Zingiber officinale Roscoe and Ocimum sanctum L., which are commonly grown in and around Mysore district, were collected during June to December, The plant species were identified taxonomically at the Department of Studies in Botany, University of Mysore (Table 3.1) : Test organisms A sick plot of downy mildew pathogen P. halstedii has been maintained in the Department of Biotechnology, University of Mysore, Mysore since the last 10 years (N 24º18, E 79º 26, 903 m altitude). In order to collect inoculum for the experiment, susceptible cv. Morden was grown in sick plot. Thirty day old infected seedlings showing profuse downy growth was selected and inoculum was harvested either late evening or early morning : 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 different solvents such as hexane, benzene, butanol, Ethyl acetate, chloroform and 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 : 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 method 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 h in moist chambers by facing abaxial surface upside at 22±1ºC in the dark. In the whole leaf method, downy mildew 36

3 infected leaves were washed to remove the existing sporangia and surface wetness was removed by blot drying. Different concentrations of the plant extract were applied on the abaxial surfaces of the leaf by coating with sterilized brush. After incubating in a moist chamber for h, the leaf discs and whole leaves were analyzed for sporulation under stereo binocular microscope and categorized as explained below. The anti-mildew activity was evaluated on following scale: Complete inhibition (+++), Moderate inhibition (++), least inhibition (+) and no inhibition (-). 3.3: Results Anti-mildew activity of medicinal plants against sunflower downy mildew Among the six solvent extracts used, methanolic extracts were found to be the best in inhibiting the sporulation of P. halstedii, whereas remaining solvent extracts were phytotoxic to the leaf. Among methanolic extracts of eight medicinal plants, M. citrifolia (fruit and bark), T. cordifolia (stem) and Z. officinale (rhizome) 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 3.2, Fig. 3.2, 3.3). Table 3.1: List of medicinal plants screened against downy mildew disease Sl.No. Medicinal plants Family Part used 1 Centella asiatica Apiaceae Leaf 2 Morinda citrifolia Rubiaceae Fruit and Bark 3 Cleodendrum inermae Verbinaceae Leaf 4 Ocimum sanctum Lamiaceae Leaf 5 Zingiber officinale Zingiberaceae Rhizome 6 Tinospora cordifolia Menispermaceae Stem 7 Eucalyptus globosus Myrtaqceae Leaf 8 Phyllanthus amarus Euphorbiaceae Leaf 37

4 38

5 Fig. 3.1: Medicinal plants used for screening the anti-mildew activity 39

6 Fig.3.2: Sunflower downy mildew disease a. Downy mildew disease symptoms b. Sporangia on the lower surface of the leaf c. Infection in the cotyledonary stage d. Sporangiophore with sporangia e. Oospore f. Sporangia observed under microscope 40

7 Fig.3.3: Anti-mildew activity of methanol extracts of selected medicinal plants on P.halstedii by leaf disc method. 41

8 Fig.3.4: Anti-mildew activity of methanol extracts of selected medicinal plants on P.halstedii by whole leaf method. 42

9 3.4 Discussion In the present study we have screened eight medicinal plant extracts for their anti-mildew activity against sunflower downy mildew disease. Among the solvent extracts, methanolic extracts showed positive result against P. halstedii. 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 activities (Pawar and papdiwal, 2010; Mohan et al., 2008 and wang et al., 2001). But the management of oomycetes phytopathogen is a challenge as it behaves differently from other fungi and is a seed borne-systemically infecting pathogen. Considering use of plant extract as ecofriendly approach to manage phytopathogens, in order 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 S. graminicola, the causative agent of pearl millet downy mildew (Deepak et al., 2005). 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 acitivty (Deepak et al., 2007). 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 methanolic extracts of 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 (Habsah et al., 2000; Mau et al.,2003 and Suhaj et al.,2006). Aloe vera which is having medicinal value was analyzed against Aspergillus flavus and Aspergillus niger for its antifungal activity. The neem seed kernel extract at 5% 43

10 concentration inhibited the sporangial germination of P. viticola, the downy mildew pathogen (Rajeshwari et al., 2008). Antimicrobial activity of fruit extract of M. citrifolia against bacterial pathogens was reported in earlier studies (Natheer et al., 2012). From the results obtained it was concluded that, methanolic extracts of M. citrifolia, (Fruit, Bark) T. cordifolia (Stem) and Z. officinale (rhizome) were the better sources for the development of ecofriendly management strategy against downy mildew disease of sunflower. Among the three, the fruit extract of M. citrifolia inhibited the sporulation at higher level. Hence further phytochemical analysis and characterization of bioactive compound was carried out. 44