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Identification and Evaluation of Antifungal Compounds From Botanicals for the Control of Sugarcane Red Rot Pathogen, Colletotrichum Falcatum

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Red rot is a devastating disease in sugarcane caused by fungus, Colletotrichum falcatum. In this study, eighteen different botanicals were screened for identifying effective antifungal compound against C. falcatum. Among the plants screened, 15 per cent aqueous leaf extract of Psoralea corylifolia alone inhibited 100 per cent growth of both mycelium as well as spore germination under in vitro conditions. The extract did not exhibit any inhibitory effect to the beneficial microbes viz., Pseudomonas fluorescens, Bacillus megaterium and Gluconacetobacter diazotrophicus which are normally used in sugarcane. The effective plant extracts exhibiting 100 per cent antifungal activity was subjected to TLC, HPLC and GC-MS analysis to identify the bioactive antifungal compound. It revealed the presence of 7H-furo [3,2-G] (1) benzopyran-7-one as main bioactive compound which is thought to be the intermediate of antifungal compound, 8 – methoxypsoralen formed during biosynthesis. Article Citation: Rajkumar D and Murugesan R. Identification and Evaluation of Antifungal Compounds from Botanicals for the Control of Sugarcane Red Rot Pathogen, Colletotrichum falcatum. Journal of Research in Agriculture (2013) 2(1): 164-172. Full Text: http://jagri.info/documents/AG0044.pdf
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  Identification and Evaluation of Antifungal Compounds from Botanicals for the Control of Sugarcane Red Rot Pathogen, Colletotrichum falcatum    Keywords: Sugarcane Red Rot,  Colletotrichum    falcatum, Psoralea corylifolia,  Antifungal Activity. ABSTRACT: Red rot is a devastating disease in sugarcane caused by fungus, Colletotrichum    falcatum . In this study, eighteen different botanicals were screened for identifying effective antifungal compound against C.    falcatum. Among the plants screened, 15 per cent aqueous leaf extract of Psoralea corylifolia  alone inhibited 100 per cent growth of both mycelium as well as spore germination under in vitro conditions. The extract did not exhibit any inhibitory effect to the beneficial microbes  viz. , Pseudomonas fluorescens , Bacillus megaterium  and Gluconacetobacter diazotrophicus  which are normally used in sugarcane. The effective plant extracts exhibiting 100 per cent antifungal activity was subjected to TLC, HPLC and GC-MS analysis to identify the bioactive antifungal compound. It revealed the presence of 7H-furo [3,2-G] (1) benzopyran-7-one as main bioactive compound which is thought to be the intermediate of antifungal compound, 8  –  methoxypsoralen formed during biosynthesis. 164-172 | JRA | 2013 | Vol 2 | No 1 This article is governed by the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which gives permission for unrestricted use, non-commercial, distribution and reproduction in all medium, provided the srcinal work is properly cited. www.jagri.info Journal of Research in Agriculture An International Scientific Research Journal Authors: Rajkumar D* and Murugesan R. Institution: Department of Agricultural Microbiology, Tamil Nadu Agricultural University, Coimbatore,  –   641 003, Tamilnadu, India. Corresponding author: Rajkumar D. Email: Web Address: http://www.jagri.info/ documents/AG0044.pdf. Dates:   Received: 11 May 2013  Accepted: 28 May 2013  Published: 13 June 2013   Article Citation: Rajkumar D and Murugesan R. Identification and Evaluation of Antifungal Compounds from Botanicals for the Control of Sugarcane Red Rot Pathogen, Colletotrichum    falcatum.   Journal of Research in Agriculture (2013) 2(1): 164-172 Original Research Journal of Research in griculture    J  o  u  r  n  a   l  o   f   R  e  s  e  a  r  c   h   i  n   A  g  r   i  c  u   l  t  u  r  e An International Scientific Research Journal    INTRODUCTION Sugarcane is one of the most important cash crops grown in India which contributes to the tune of 7.5 per cent of agricultural production of the country (Viswanathan, 2010). But, sugarcane red rot disease caused by fungus Colletotrichum falcatum  is considered as very serious disease and it was responsible for the elimination of many elite sugarcane varieties such as Co 312, Co 658, Co 997, Co 1148, CoC 671 etc. Upto 100  per cent yield loss has been reported in severe epiphytotics (Siddique et al.,  1983). The use of chemical fungicide bavistin is the common practice followed by the sugarcane growers for the control of sugarcane red rot. Practicing sett treatment with systemic fungicides alone is not sufficient to protect such a long duration crop. The use of synthetic fungicide leads to several  problems such as residue in food and feed, pathogen resistance, toxicity to non target organism and environmental pollution (Angelo et al.  2012). In addition to these, elimination of soil born inoculum through chemicals is difficult and costly. Development of resistant varieties through breeding methods is a long term endeavour. Therefore, with the increasing public awareness of environmental safety and persistent demand for ecofriendly products, we are forced to  produce quality products both for export and domestic consumption. Hence, an alternative approach is the use of botanicals in management of this disease which are eco friendly in addressing the problem. MATERIALS AND METHODS Plants screened for antifungal study Eighteen plants belonging to 14 families were taken for screening antifungal activity against Colletotrichum falcatum.  All the plants were collected from Medicinal Plant Section maintained at Botanical Garden and agricultural fields of Tamil Nadu Agricultural University, Coimbatore (Table 1). All the experiments were done with three replications and the data given are the mean of three replications. Since this study is a preliminary qualitative screening study under in vitro  conditions quantitative data were not given and so statistics was also not applied for the data. Microbial cultures taken for study Sugarcane red rot fungi, Colletotrichum falcatum  was obtained from Department of Plant Pathology, Sugarcane Breeding Institute, ICAR, Coimbatore. Three beneficial microorganisms viz.,    Pseudomonas    fluorescens,    Bacillus   megaterium  and Gluconacetobacter    diazotrophicus  commonly applied for sugarcane crop were obtained from Department of Agricultural Microbiology, Tamil Nadu Agricultural University, Coimbatore. Preparation of plant extracts Fresh plant parts of all botanicals were thoroughly washed with running tap water followed by rinsing twice with distilled water. The plant materials were shade dried for 1 h. Weighed plant materials were homogenized in mixer grinder with distilled water (1:1 w/v). The slurry of plant extracts obtained was passed through glass wool and the filtrate was then centrifuged at 5000 rpm for 10 min. Supernatant of botanicals thus obtained were the standard aqueous plant extract solution (100  per cent). For methanol extract, weighed plant materials were homogenized in mixer grinder with methanol (1:3 w/v). The slurry samples collected in a beaker were covered with aluminium foil and incubated in refrigerator at 4°C for 24 h. After incubation period, slurry was filtered using glass wool and subsequently with Whatman No. 1 filter paper. The clear filtrate was allowed to evaporate in petriplates and the residues were dissolved in distilled water to obtain standard methanol  plant extract solution of all botanicals (Biri Singh, 2004).  In vitro  testing of plant extracts against test pathogens The antifungal effects of all the plant extracts were screened by poison food technique (Horsefall, 1956). An appropriate amount of plant extracts were added to sterilized potato dextrose agar (PDA) medium 165 Journal of Research in Agriculture (2013) 2(1): 164-172 Rajkumar and Murugesan , 2013  to obtain the desired concentration of 25 per cent. Then the medium containing plant extracts were kept in water  bath at 45°C for 60 min to eliminate bacterial contamination (Martinez et al  ., 1990) and plated after cooling it to luke warm temperature. C. falcatum  mycelial disc of 9 mm diameter was taken from 3-5 days old culture plate and  placed to the centre of each plates and incubated at 28°C. After 5 days of incubation the diameter of mycelial colony was measured and the per cent inhibition for all botanicals was calculated by using the formula (Vincent, 1927). Where, C and T were mycelial growth (cm) of test fungi in control and treatments, respectively. All treatments were done in triplicates and average values were taken for interpretation. Minimum inhibitory concentration (MIC) Minimum inhibitory concentration is the lowest concentration at which 100 per cent inhibition of mycelial growth is obtained. Among the different  botanicals screened,  P. corylifolia  aqueous leaf extract, which exhibited effective inhibition over C. falcatum , was tested at different concentrations (5, 10, 15 and 20 per cent) to find out MIC by poison food technique.  Evaluation of the effect of  Psoralea   corylifolia  extract   on the spore germination of Colletotrichum falcatum    P.   corylifolia  extract of different concentrations (5, 10, 15 and 20 per cent) were prepared in tubes for checking its effect on spore germination of C.    falcatum . A loopful of conidia from sporulated culture in oatmeal enriched PDA medium were transferred into extract containing tubes. From each extract concentration, 100 μ l was taken into the cavity slides along with the control (without extract). These slides were kept in a moist chamber for the period of 24 h for germination of spores and the effect of  P. corylifolia  extract on spore germination was determined by counting germinated spores under microscope and it was compared with the control (Jayakumar, 2007). Evaluation of the effect of  P. corylifolia  extract on growth pattern of beneficial organisms  In vitro  testing of the  P.   corylifolia  plant extract was carried out on beneficial microflora to assess its influence on their growth. Twenty four hour old cultures of  Pseudomonas    fluorescens,  and  Bacillus   megaterium  were streaked on the nutrient agar medium containing specific plant extract and Journal of Research in Agriculture (2013) 2(1): 164-172 166 Rajkumar and Murugesan , 2013 Table 1. List of medicinal plants screened for its antifungal activity S.No   Family  Botanical name Parts used  1. Amaranthaceae  Achyranthes aspera  Leaf 2. Malvaceae  Abelmoschus esculentus  Leaf 3. Papilionaceae  Abrus precatorius  Leaf 4. Annonaceae  Annona squamosa  Leaf 5. Nyctaginaceae  Bougainvillea spectabilis  Leaf 6. Asclepiadaceae Calotropis gigantea  Leaf 7. Verbenaceae Clerodendrum inerme  Leaf 8. Caesalpiniaceae Caesalpinia bonduc  Bark 9. Sapindaceae Cardiospermum halicacabum  Leaf 10. Asteraceae  Eclipta alba  Leaf 11. Verbenaceae  Lantana camara  Leaf 12. Moringaceae  Moringa oleifera  Leaf 13. Meliaceae  Melia azadirachta  Leaf 14. Fabaceae  Prosopis juliflora  Leaf, Stem 15. Papilionaceae  Psoralea corylifolia  Leaf, Stem 16. Apiaceae Trachyspermum ammi  Leaf, Stem 17. Verbenaceae Vitex negundo  Leaf 18. Asteraceae Wedelia urticaefolia  Leaf Inhibition (%) = (C  –   T) X 100 C    Gluconacetobacter    diazotrophicus  was streaked on the  potato dextrose agar medium containing plant extract and suitable control (without plant extracts) was also maintained. The plates inoculated with  Pseudomonas    fluorescens,  and  Bacillus   megaterium  were incubated for two days whereas Gluconacetobacter    diazotrophicus  plates were incubated for five days. The growth of the organism was observed visually, compared with that of the control and given the remark as  –   for no growth, + for very less growth, ++ for moderate growth and +++ for growth equivalent to control. Control indicates petri plates containing PDA medium without plant extracts. It was taken to check whether there is any difference in the growth pattern of beneficial microbes inoculated in PDA medium with plant extracts and without  plant extracts. Identification of the fungitoxic principles through TLC, HPLC and GC-MS analysis  Thin layer chromatographic study was carried out to identify the nature of active principle in the effective plant extracts. The solvent systems used were ethyl acetate: methanol: ammonia solution (17:5:1), chloroform: acetic acid (9:1) and ethyl acetate: formic acid: acetic acid: water (100:11:11:27) for alkaloids,  phenols and flavonoids, respectively.   Dragendroff’s reagent, Folin-Ciocalteu reagent and Diphenylboryloxyethylamine (1 per cent) with 5 per cent  polyethylene glycol 4000 were used as spray reagents. The dried TLC plate was exposed to UV light (254 nm) and observed for fluorescent spots. The compounds separated in TLC were checked for its antifungal activity against C. falcatum  by poison food technique . The active compounds which exerted antifungal activity were subjected to HPLC analysis. HPLC analysis HPLC  –   shimadzu model L 6200 (Varian) diode array detector in combination with Beckman ultrasphere supelco ODS column (250 x 4.6 mm) was used for analysis. The analysis was performed at a flow rate of 0.35ml min -1  using Methanol: Water (40:60) as mobile  phase. The UV detector was set at 260nm wavelength for  psoralen (Murali et al.  2002). Psoralen standard solutions (0.5, 1.0, 1.5 and 2.0 percent) were prepared and 20 µl of the solution was injected to record the retention time. The individual peaks separated in HPLC was collected through fraction collector and evaluated for its antifungal activity by poison food technique. The peak which exerted antifungal activity was again subjected to GC-MS analysis. GC-MS analysis GC-MS JEOL GC mate  –   Hitachi model L 6200 (Varian) photo array detector in combination with Beckman Ultrasphere supelco ODS column (250 x 4.6 mm) was used for analysis. The analysis was performed at a flow rate of 0.35ml min -1 . The UV detector was set at 260nm wavelength for Psoralen. The peaks obtained in chromatogram were analysed for its mass spectrum to identify the compounds by comparing its molecular weight with the compounds present in MS library. RESULTS AND DISCUSSION Antifungal activity of different botanicals Among 18 plants tested for the antifungal activity against C. falcatum,  15 per cent aqueous leaf extract of  P. corylifolia  inhibited 100 per cent growth of  both mycelium as well as spore germination under laboratory conditions (Plate 1and 2). None of the plant extracts other than  P. corylifolia  were able to exhibit comparatively appreciable inhibition over C. falcatum . The presence of antifungal compounds in medicinal  plants has long been recognized as an important factor to disease resistance (Mahadevan, 1982; Kurucheve et al. , 1997). Such compounds, being biodegradable and selective in their toxicity are considered valuable for controlling different plant diseases (Singh and Dwivedi, 1987). Rajendra Prasad et al.,  (2004) reported the antifungal activity of extracts obtained from  P. corylifolia  seeds against dermatophytic fungi such as Trichophyton rubrum , Trichophyton mentagrophytes ,  Epidermophyton 167 Journal of Research in Agriculture (2013) 2(1): 164-172 Rajkumar and Murugesan , 2013

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