Biological Control 57 (2011) 175–183 Contents lists available at ScienceDirect Biological Control journal homepage: www.elsevier.com/locate/ybcon Combined application of botanical formulations and biocontrol agents for the management of Fusarium oxysporum f. sp. cubense (Foc) causing Fusarium wilt in banana R. Akila ⇑, L. Rajendran, S. Harish, K. Saveetha, T. Raguchander, R. Samiyappan Department of Plant Pathology, Centre for Plant Protection Studies, Tamil Nadu Agricultural University, Lawley Road, Coimbatore 641 003, TamilNadu, India a r t i c l e i n f o Article history: Received 10 August 2009 Accepted 22 February 2011 Available online 6 March 2011 Keywords: Fusarium wilt Fusarium oxysporum f. sp. cubense Datura metel Peroxidase Polyphenol oxidase Pseudomonas fluorescens 1 Bacillus subtilis a b s t r a c t Plant products along with biocontrol agents were tested against Fusarium wilt of banana caused by Fusarium oxysporum f. sp. cubense (Foc). Of the 22 plant species tested, the leaf extract of Datura metel (10%) showed complete inhibition of the mycelial growth of Foc. Two botanical fungicides, Wanis 20 EC and Damet 50 EC along with selected PGPR strains with known biocontrol activity, Pseudomonas fluorescens 1, Pf1 and Bacillus subtilis, TRC 54 were tested individually and in combination for the management of Fusarium wilt under greenhouse and field conditions. Combined application of botanical formulation and biocontrol agents (Wanis 20 EC + Pf1 + TRC 54) reduced the wilt incidence significantly under greenhouse (64%) and field conditions (75%). Reduction in disease incidence was positively correlated with the induction of defense-related enzymes peroxidase (PO) and polyphenol oxidase (PPO). Three antifungal compounds (two glycosides and one ester) in D. metel were separated and identified using TLC, RP-HPLC (Reverse Phase-High Pressure Liquid Chromatography) and mass spectrometry. In this study it is clear that combined application of botanical formulations and biocontrol agents can be very effective in the management of Fusarium wilt of banana. Ó 2011 Elsevier Inc. All rights reserved. 1. Introduction Banana (Musa sp.) is one of the oldest fruits known to mankind. It is considered as the fourth most widely consumed food crop in the world after rice, wheat and corn based on gross value (http:// www.suite101.com). India ranks first in terms of production accounting for 26 million MT (http://faostat.fao.org). Among the various constraints affecting banana cultivation, Fusarium wilt caused by Fusarium oxysporum f. sp. cubense (E.F. Smith) Snyder and Hansen (Foc) is considered one of the most important threats in Asia, Africa, Australia and tropical America (Hwang and Ko, 2004). In Tamil Nadu, India, the disease was highly destructive on the apple flavor cv. Rasthali (Lakshmanan et al., 1987). At present the disease is widespread in almost all the banana growing regions of India and varieties like Rasthali (AAB) and Virupakshi (AAB) are highly susceptible to this disease and threatened with extinction (Thangavelu et al., 2001). Fusarium wilt is a classic vascular wilt disease in which the fungus occludes the xylem vessels causing water blockage. It survives in soil for long periods and thus susceptible genotypes cannot be grown in an infested field for up to 30 years (Ploetz, 2000). The symptoms become evident after 5–6 months of planting and are expressed both externally and ⇑ Corresponding author. E-mail address: akilpatho@gmail.com (R. Akila). 1049-9644/$ - see front matter Ó 2011 Elsevier Inc. All rights reserved. doi:10.1016/j.biocontrol.2011.02.010 internally. The disease causes yellowing of leaf margin of oldest leaves, hanging of leaves around pseudostem, splitting of pseudostem and yield losses in the later stages. Plants affected by wilt generally produce unmarketable bunches and the disease ultimately destroys the entire plant. Various control measures have been practised to manage this disease, including destruction of diseased plants, sanitary measures, use of disease-free tissue culture planting material, use of tolerant variety and other integrated management methods. Chemicals are also widely utilized for the management of this disease. However, indiscriminate use of chemicals is known to cause health hazards to human beings besides warranting repeated application. As an alternative approach, biocontrol agents are being used for the management of various diseases (Kavino et al., 2008; Harish et al., 2009a). Botanicals with antifungal compounds have been identified and these can be exploited for the management of diseases (Kagale et al., 2004). Botanicals have low mammalian toxicity, target specificity, biodegradability and contain many active ingredients in low concentrations, thus possess biocidal activity against several insect pests and pathogens (Harish et al., 2008; Kalaycioglu et al., 1997). Kagale and coworkers (2004) documented that the methanolic extract of Datura metel exhibited 85% reduction of mycelial growth of Rhizoctonia solani. In addition, aqueous leaf extract of D. metel was known for its antifungal activity against late leaf spot and rust pathogens, Phaeoisariopsis personata and 176 R. Akila et al. / Biological Control 57 (2011) 175–183 Puccinia arachidis, respectively (Kishore and Pande, 2005). Hence the antifungal compounds from D. metel can be utilized for the management of plant diseases. Wanis is a commercial botanical fungicide developed and marketed by Southern Petrochemical Industries (SPIC) Ltd. The major active component of the botanical fungicide consists of monoterpenoids from herbal oils. Wanis 40 EC (v/v) showed antifungal activity against Fusarium solani, Fusarium equiseti, F. oxysporum, Phytophthora capsici, Sclerotinia sclerotiorum, Pyricularia oryzae, Drechslera oryzae and R. solani (Narasimhan et al., 1998; Narasimhan et al., 1999). Plants possess various inducible defense mechanisms to protect themselves against pathogen attack (Pieterse et al., 1998). The inducers include pathogens (Block et al., 2005), Plant Growth Promoting Rhizobacteria (PGPR) (Saravankumar et al., 2007), chemicals (Michael et al., 2001) and botanicals (Harish et al., 2008). Induced systemic resistance (ISR) develops as a result of colonization of plant roots by PGPR. Fluorescent pseudomonads are well known for their ability to colonize the root tissues of a wide range of crop plants and promote plant growth by the production of secondary metabolites and volatiles and inducing enzymes (Stougard, 2000; Han et al., 2006). Fluorescent pseudomonads are among the most effective rhizosphere bacteria, because in addition to disease control, they exert beneficial effect on plant growth promotion (Kloepper et al., 1980). ISR elicited by PGPR has shown promise in managing a wide spectrum of plant pathogens in several plant species under greenhouse and field environments (Radjacommare et al., 2004; Zehnder et al., 2000; Murphy et al., 2003). Phytohormones produced by PGPR play a major role in growth promotion and many bacteria have the ability to produce auxins, gibberellins, cytokinins and ethylene (García de Salamone et al., 2001; Remans et al., 2008). Besides promoting growth, PGPR induce defense related proteins and enzymes which can provide resistance against plant diseases (Nandakumar et al., 2001). It is the need of the day to find an alternate approach for the management of Fusarium wilt of banana. Hence in this study, an integrated approach was made to manage Fusarium wilt of banana using botanical bioformulations and biocontrol agents. 2. Materials and methods 2.1. Plant materials, pathogen and plant extract Banana cv. Rasthali and the F. oxysporum f. sp. cubense (Foc) isolate (Race 1 – obtained from NRCB, National Research Centre for Banana, Trichy, Tamil Nadu) were used in this study. Twenty-two plant species with proven antifungal activity were selected for further experiments (Table 1). Twenty-five gram of fresh leaves and rhizome of turmeric were collected manually and extracted with 25 ml of sterile water (1 g/ml, w/v) using pestle and mortar. The extract was filtered through muslin cloth and finally through Whatman No. 1 filter paper and filter sterilized using Seitz filter (45 lm). This formed the standard plant extract solution (100%) (Shekhawat and Prasada, 1971). Ten milliliter of the standard plant extract solution (100%) was mixed with 90 ml of the sterilized Potato Dextrose Agar (PDA) medium to get the required concentration (10%) of the plant extract. Twenty ml of this mixture was poured into sterilized Petri dishes and allowed to set. A nine mm actively growing PDA culture disc of Foc was placed at the center of the medium. The plates were incubated at room temperature (28 ± 2 °C) for seven days. PDA without plant extract served as control. Three replications were maintained for each treatment (Schmitz, 1930). The mean diameter of the mycelial growth of the pathogen was recorded and the results were expressed as per cent reduction of mycelium growth over that of the control (Vincent, 1927). 2.2. Extraction of antifungal compounds from D. metel Antifungal activity of D. metel was demonstrated by various authors (Kagale et al., 2004; Kishore and Pandey, 2005). Leaves of D. metel were homogenized in different solvents (1:1 w/v) viz., methanol, acetone, diethyl ether, ethyl acetate and chloroform separately. Extracts were filtered through two layers of muslin cloth and centrifuged at 7500g for 20 min. Solvent phase of the extracts were evaporated in vacuum at 40 °C using a Rotavapor R-114 (Bucchi) and the residue obtained was dissolved in 2 ml of sterile distilled water. The antifungal nature of various solvent extracts was tested against the mycelial growth of Foc by inhibition zone technique (Bauer et al., 1966). 2.3. Botanical fungicide formulation The botanical fungicide Damet 50 EC is prepared in Plant Pathology laboratory, TNAU, Coimbatore, Tamil Nadu. The method of preparation is as follows. Two kg of D. metel leaves were washed and ground with methanol (1:2) and filtered several times through cheese cloth and the supernatant solution was collected. The clear solution was condensed in vacuum and reduced to half the volume. Fifty EC formulation was developed using this condensed, partially purified extract (100% concentration) by adding the recommended quantities of emulsifying agent (Unitox 30x and 60y), stabilizing agent (Epichlorohydrin) and solvent (Cyclohexanone) and named as Damet 50 EC. 2.4. Testing the bioefficacy of Wanis and biocontrol agents The antifungal activity of Wanis at various concentrations (0.25, 0.50, 0.75 and 1.0%) was tested against the mycelial growth of Foc by poisoned food technique (Shekhawat and Prasada, 1971). Two bacterial antagonists Pf1 and TRC 54 were used in this study. Pf1 is an isolate of Pseudomonas fluorescens that has been used against most of the diseases of rice, ragi, chickpea, tomato, chilli, banana and mango etc., all over India since 1995 (Vidhyasekaran and Muthamilan, 1995). Its antifungal activity has been well-documented by several authors (Radjacommare et al., 2004; Ramamoorthy et al., 2002). TRC 54 which has been identified as Bacillus subtilis by ITS amplification (NCBI Accession number EF141519) was isolated from banana rhizosphere, Tamil Nadu, India. This strain was selected after testing its growth promoting activity by roll towel and potculture experiments on rice. These two biocontrol isolates were tested for their inhibitory activity against the mycelial growth of Foc using dual plate technique (Dennis and Webster, 1971). A 9 mm dia culture disc of Foc was placed on PDA medium at one side of Petri plate. Six days after placing the mycelial disc, bacterial strains were streaked on the opposite side. The plates were incubated at room temperature for 10 days. Mycelial growth and inhibition zone (mm) of the pathogen were measured. A talc based formulation of the biocontrol agents was prepared as per the procedure described by Nandakumar et al. (2001). 2.5. Compatibility among biocontrol agents, D. metel extract and Wanis Methanolic extract of D. metel was prepared (1 g/ml, w/v), evaporated to dryness, dissolved in sterile water, filter sterilized using a Seitz filter. This extract contained the active components of D. metel. Three sterile paper discs were placed at equidistance to each other on the bacteria seeded medium and numbered serially as 1, 2 and 3. Fifty microliter of concentrated active principles in the sterile water medium was added to the first sterile filter paper disc. Similarly, 50 ll of 100 ppm streptomycin sulfate and sterile R. Akila et al. / Biological Control 57 (2011) 175–183 Table 1 Effect of plant extracts (10%) on the mycelial growth of Fusarium oxysporum f. sp. cubense. S. No 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 Scientific name Diameter of mycelium (cm)* Per cent reduction over control Ageratum conizoides Annona squamosa Azadirachta indica Allium sativum x A. Ceba Calotropis procera Coleus forskohlii Curcuma longa Catharanthus roseus L Datura metel Eclipta alba Eucalyptus globulus (Labill) Lawsonia inermis L Lantana camera Nerium odorum Ocimum sanctum Psoralea corylifolia Polyalthia longifolia Ricinus communis Tecoma grandis Thevetia peruviana Vitex negundo L Andrographis paniculata Control (without plant extract) 4.17cd 6.50hi 6.50hi 6.67i 52.07 25.30 25.30 23.33 5.30efg 3.20b 2.77b 5.97gh 0.90ª 4.00c 6.50hi 39.10 63.22 68.20 31.38 89.66 54.02 25.30 2.83b 5.10ef 4.67de 5.63fg 4.50cd 4.33cd 6.90i 7.00i 5.50fg 5.30efg 7.00i 67.50 41.40 46.32 35.30 48.28 50.22 20.70 19.54 36.80 39.00 19.54 8.70j - * Mean of three replications. Means followed by common letter are not significantly different at 5% level by DMRT. water were added to the 2nd and 3rd filter paper discs as chemical check and control, respectively. The plates were incubated at room temperature for 8 days and observed for the zone of inhibition. Absence of inhibition zone around the disc indicated compatibility with respective bacterial isolates and the presence of inhibition zone indicated incompatibility. Similarly, Wanis 20 EC (1%) was tested for compatibility with Pf1 and TRC 54. The compatibility between the bacterial isolates was tested by streaking them opposite to each other in nutrient agar plate and observing either overgrowth or the inhibition zone (Fukui et al., 1994). 2.6. Efficacy of bioagents against Foc under greenhouse conditions Healthy banana suckers of the cv. Rasthali obtained from a wiltfree banana garden were used for the experiments. Normal recommended dose of fertilizers and Farm Yard Manure were applied to each pot. Farm Yard Manure (FYM) is a decomposed mixture of dung and urine of farm animals along with waste feeds, fodder and litter. Average nutrient content (%) of FYM is Nitrogen 0.5– 1.5, Phosphorus 0.4–0.8 and Potassium 0.5–1.9. When the plants reached their second month of growth, they were inoculated by corm injection of a spore suspension of the pathogen (3 ml/plant, 106 cfu/ml) and sand maize inoculum (10 per cent w/w/kg soil) (Saravanan, 2002). Treatments were replicated three times in Completely Randomized Design. Each replication consisted of one plantain. Treatments were applied on the 2nd, 4th, 6th and 8th month after planting. 2.7. Field experiment A field experiment was laid out in Tamirabarani belt of Thoothukudi district (Tamil Nadu, India). The banana field selected was heavily infested with Fusarium wilt for the past 5 years. The experiment was laid out in a randomized complete block design 177 (RBD) with twelve treatments replicated three times. In each treatment, there were six plants per replication. The banana suckers of cultivar Rasthali without Fusarium wilt infection was obtained from the wilt-free garden. The treatments and the schedule of application were the same as that in the greenhouse experiment. A capsule filled with carbendazim (60 mg/capsule) was applied by the corm injector designed at TNAU. The capsule was applied to the holes made at an angle of 45° diagonally in the corm. Observations on the incidence of wilt disease was scored based on a 1–5 scale (Ploetz et al., 1999). The percent wilt index was worked out using Mc Kinney’s (1923) formula. Besides disease incidence, observations of growth parameters such as girth and height of pseudostem and leaf area were recorded according to the guidelines of the International Institute for the improvement of Banana and Plantain (INIBAP). 2.8. Assay of defense related enzymes The banana root tissues from different treatments were collected and homogenized immediately with 2 ml of Sodium phosphate buffer (0.1 M, pH 7.0) at 4 °C for the assay of PO and PPO activity. The homogenate was centrifuged for 20 min at 10,000 rpm. For the enzyme assay, samples were collected from 5 to 6 month old plants, on 0th, 3rd, 6th, 9th and 12th day after the application of treatments. The changes in peroxidase and polyphenol oxidase activities were determined by colorimetric assays described by Hammerschmidt et al. (1982) and Mayer et al. (1965). The expression pattern of different isoforms of peroxidase and polyphenol oxidase in different treatments was studied through activity gel electrophoresis based on the protocol described by Sindhu et al. (1984) and Jayaraman et al. (1987), respectively. 2.9. Characterization of antifungal compounds in D. metel using TLC, RP-HPLC and mass spectrometry Methanolic extract (100%) of D. metel was evaporated to dryness in flash evaporator and the active principles were dissolved in 1 ml methanol. Using the capillary tube, 20 ll of the extract was applied on the activated plates and run separately for 90 min in the solvent system of chloroform: acetic acid (9:1). Phenolic compounds were detected by spraying Folin–ciocalteau reagent (1 N) followed by spraying 20% Na2CO3 solution (Sadasivam and Manickam, 1992). Presence of phenols is indicated by blue spots. The relation to front (Rf) of the spots was calculated by measuring the distance moved by the solute from the origin and dividing it by the distance (cm) moved by the solvent from the origin. Phenolic compounds in TLC purified retention factors (Rf1, Rf2 and Rf3) were separated and identified using Shimadzu LC 8A RP-HPLC with C18 column. The pressure maximum was set to 300 psi with the flow rate of 1 ml/min. UV wave length used for detection was 280 nm. Two pumps, consisting of (A) acetonitrile and (B) 0.1% phosphoric acid were used to run the TLC purified phenolic compounds. Gallic acid, vanillic acid, caffeic acid, syringic acid, coumarin, cinnamic acid, 8-hydroxy quinoline and flavone were chosen as standards. The ESI-LC–MS of the TLC purified sample was recorded on a MICROMASS QUATTRO II triple quadrupole mass spectrometer having a JASCO PU-980 HPLC pump which is available at the Regional Sophisticated Instrumentation Centre, Central Drug Research Institute, Lucknow, India. The column was WATER SPHERISORB ODS 2 (250  4.6 mm  5 l). The solvent was acetonitrile:water +0.1% formic acid. Gradient elution at 1.0 ml/min. The photodiode array was monitored at 200–650 nm and recorded at 220 nm. The mass spectra were scanned in the range 80–1000 DA in 2.5 s. The ESI capillary was set at 3.5 kv and the cone voltage was 40 V. Dry 178 R. Akila et al. / Biological Control 57 (2011) 175–183 nitrogen was used as the nebulizer (10 lit per h) and drying gas (250 l/h). The source temperature was 90 °C. Table 3 Effect of PGPR strains on the mycelial growth of Fusarium oxysporum f. sp. cubense. PGPR strains 2.10. Statistical analyses The data were statistically analyzed (Rangasamy, 1995) using the IRRISTAT version 92 developed by the International Rice Research Institute Biometrics unit, the Philippines (Gomez and Gomez, 1984). Lab experiments were carried out under Completely Randomized Block Design (CRD) and field trials were conducted using Randomized Block Design (RBD). The percentage values of the disease index were arcsine transformed. Data were subjected to analysis of variance (ANOVA) and means were compared by Duncan’s Multiple Range Test (DMRT). 3. Results 3.1. Efficacy of plant products, Wanis and biocontrol agents against F. oxysporum f. sp. cubense The leaf extract of D. metel exhibited the maximum reduction (89.66%) of the mycelial growth of the pathogen. This was followed by Curcuma longa, Lawsonia inermis and Coleus forskohlii which were comparable with each other (Table 1). Methanol extract reduced the mycelial growth significantly (1.17 cm inhibition zone) from other solvent extracts. This was followed by chloroform extract (0.3 cm inhibition zone) which was on par with ethyl acetate and acetone (Table 2). All the above solvents when used alone did not show any inhibitory activity. So, it is concluded that the antifungal compounds were highly soluble in methanol. Wanis at two different concentrations (0.75% and 1.0%) were highly effective in inhibiting the mycelial growth of Foc (data not shown). The strain Pf1 exhibited maximum inhibition of the mycelial growth which was clearly discerned by complete absence of fungal mycelium in the inhibition zone surrounding the bacterial colony (Table 3). Results of the compatibility test indicated that the active principles of D. metel were compatible with Pf1 and TRC 54. Wanis 20 EC was also not toxic to Pf1 and TRC 54. Compatibility test between the biocontrol agents indicated that there was no antagonistic effect between the bacterial isolates (Pf1 and TRC 54). As they were compatible, the formulation of individual strains were combined in 1:1 ratio and used for further studies (data not shown). 3.2. Evaluation of bioagents against Fusarium wilt under greenhouse and field conditions All botanical and biological treatments significantly reduced the incidence of Fusarium wilt under greenhouse conditions. Damet 50 EC alone reduced wilt by only 9%, but Pf1 and TRC 54, alone or together, reduced disease by 37–45%. Combinations of the botanical formulations with the biocontrol agents resulted in significant control of the disease. The combination of Damet 50 EC or Wanis 20 EC Table 2 Effect of various solvent extracts of Datura metel on the mycelial growth of Fusarium oxysporum f. sp. cubense. * Solvent extracts of D. metel Inhibition zone (cm)* Methanol Chloroform Ethyl acetate Diethyl ether Acetone Sterile water 1.17a 0.30b 0.13bc 0.10c 0.13bc 0.00e Mean of five replications. Means followed by a common letter are not significantly different at 5% level by DMRT. Pf1 TRC 54 MDU 63 Control Diameter of mycelium (cm)* a Inhibition zone (cm) 0.80a 0.70b 0.60c 0.00d 5.00 5.70a 6.00a 9.00b * Mean of five replications. Means followed by common letter are not significantly different at 5% level by DMRT. with both biocontrol agents revealed greatest disease reduction of all treatments (>63% reduction). Plants treated with carbendazim exhibited 45% reduction of Fusarium wilt compared with control. Considering the plant growth promotion, Wanis 20 EC + Pf1 + TRC 54 significantly increased the plant growth parameters such as height, pseudostem girth and leaf area (143.76 cm, 18.00 cm and 1180 cm2) compared to healthy control which recorded 100.60 cm, 12.33 cm and 727.20 cm2, respectively (Table 4). In the field experiment, application of Wanis 20 EC reduced wilt by 61.70%, but Pf1 and TRC 54 alone or together reduced disease by 51–65%. Among the different combinations, Wanis 20 EC + Pf1 + TRC 54 produced the greatest suppression of wilt disease (75.16%) followed by Damet 50 EC + Pf1 + TRC 54 (67.80%). Plants treated with carbendazim exhibited 64% reduction of Fusarium wilt. The yield recorded in the plot applied with the combination (Wanis 20 EC + Pf1 + TRC 54) was 33.82 t/ha, which was 11-fold higher than control (3.15 t/ha) (Table 5). The plant growth attributes plant height, pseudostem girth and leaf area were maximum in the Wanis 20 EC + Pf1 + TRC 54 applied plot (580.00 cm, 81.95 cm and 9165.00 cm2, respectively) compared to the untreated check (400.00 cm, 63.54 cm and 5048.00 cm2, respectively). 3.3. Induction of defense enzymes against Fusarium wilt Accumulation of defense enzymes was significantly higher in banana plants treated with the combination of botanical formulations and biocontrol agents compared to single treatment. PO activity started increasing from the third day after application Table 4 Effect of botanical formulations and biocontrol agents on Fusarium wilt incidence under green-house conditions. Treatments Plant height (cm)* Pseudo stem girth (cm)* Leaf area (sq cm)* Per cent wilt index (%)* Per cent reduction over control Pf1 TRC 54 Pf1 + TRC 54 Damet Damet + Pf1 Damet + TRC 54 Damet + Pf1 + TRC 54 Wanis Wanis + Pf1 + TRC 54 Carbendazim Healthy control Inoculated control 119.33abc 112.83a–d 122.33abc 101.00bcd 107.33bcd 105.33bcd 13.70bcd 13.30bcd 14.73abc 13.00cd 15.30abc 14.40bc 849.58g 833.62h 1013.88b 805.28i 873.60e 854.44f 40.13d (39.31) 46.13e (42.78) 40.03d (39.25) 66.67f (54.74) 33.19c (35.18) 33.48c (35.35) 45.27 37.09 45.41 9.08 54.74 54.34 122.87abc 16.67ab 941.60c 26.67b (31.02) 63.63 ab 135.00 143.76a abc 15.33 18.00a d 924.00 1180.00a 46.52 (43.01) 26.56b (31.02) 36.56 63.78 104.00bcd 100.6cd 13.37bcd 12.33cd 744.60j 727.20k 40.18d (39.34) 0.00a (0.36) 45.21 – 10.40d 492.32l 73.33g (58.92) 85.00d e – * Mean of three replications. Data in parentheses are arcsine transformed values. Means followed by common letter are not significantly different at 5% level by DMRT. 179 R. Akila et al. / Biological Control 57 (2011) 175–183 Table 5 Effect of botanical formulations and biocontrol agents on Fusarium wilt incidence under field conditions. * Treatments Plant height (cm)* Pseudostem girth (cm)* Leaf area (sq cm)* Per cent wilt index (%)* Per cent reduction over control No. of fruits/ hand Yield (t/ ha) Pf1 TRC 54 Pf1 + TRC 54 Damet Damet + Pf1 Damet + TRC 54 Damet + Pf1 + TRC 54 Wanis Wanis + Pf1 + TRC 54 Carbendazim Control 467.58g 453.72h 537.00c 430.00i 540.00c 525.00d 555.00b 73.50cd 72.75cd 75.50bc 67.25e 72.50cd 71.00bc 78.36ab 6114.10g 6048.00h 7661.33d 5532.00i 6669.00e 6204.00f 7708.81c 43.18f (41.08) 45.73g (42.55) 32.52c (34.77) 50.98h (45.56) 40.64e (39.61) 42.64f (40.77) 30.12b (33.28) 53.84 51.11 65.23 45.50 56.55 54.42 67.80 15.00d 13.00f 16.00c 12.00g 15.00d 14.00e 15.00d 24.30e 23.00ef 29.40bc 20.47f 25.45de 24.50e 30.51b 510.00e 580.00a 72.86cd 81.95a 7779.20b 9165.00a 35.83d (36.77) 23.24a (28.82) 61.70 75.16 17.00b 18.00a 27.70cd 33.82a 485.00f 400.00j 70.30de 63.54f 5467.20j 5048.00k 33.67c (35.47) 93.54i (75.29) 64.00 - 13.00f 10.00h 28.80bc 3.15g Mean of three replications. Data in parentheses are arcsine transformed values. Means followed by common letter are not significantly different at 5% level by DMRT. observed in plants treated with Wanis 20 EC + Pf1 + TRC 54, while inoculated control produced less induction. Similar to PO, expression of PPO was also maximum on 9th day after application of the treatments (Fig. 1B). and reached its peak on the 9th day and then started declining in the roots. Plants treated with Wanis 20 EC + Pf1 + TRC 54 resulted in maximum activity (Fig. 1A). Significant induction of PPO activity (1.340-fold increase in absorbance/min/g of root tissue) was A SD 0.17 0.8 SD 0.16 0.7 0.6 SD 0.088 0.5 SD 0.037 0.4 0.3 SD 0.016 0.2 0.1 0 0 3 6 9 Pf1+ Bacillus 54 Damet+Pf1+ Bacillus 54 Wanis Carbendazim Healthy control Inoculated control B 1.6 12 Wanis+Pf1+ Bacillus 54 SD 0.367 1.4 SD 0.356 1.2 1 0.8 0.6 SD 0.012 SD 0.078 SD 0.153 0.4 0.2 0 0 3 6 9 Pf1+ Bacillus 54 Damet+Pf1+ Bacillus 54 Wanis Carbendazim Healthy control Inoculated control 12 Wanis+Pf1+ Bacillus 54 Fig. 1. Effect of botanical formulations and biocontrol agents on (A) peroxidase and (B) Polyphenol oxidase activity in banana root. 180 R. Akila et al. / Biological Control 57 (2011) 175–183 3.4. Native gel electrophoresis The roots of treated plants expressed 3 PO isoforms, PO1, PO2 and PO3. The isozyme PO1 was observed only in the plants treated with Damet 50 EC + Pf1 + TRC 54 and Wanis 20 EC. In roots, the isoform, PPO3 appeared intensively in the plants treated with Pf1 + TRC 54 and Wanis 20 EC + Pf1 + TRC 54. Intensity of the isozyme was lesser in healthy control (Fig. 2). 3.5. Characterization of antifungal compounds in D. metel using TLC, RP-HPLC and mass spectrometry Three spots with Rf (Relation to front) values of 0.20, 0.16 and 0.10 were noted indicating the presence of phenolic compounds (Table 6). Partially purified phenolics from Rf1 and Rf2 exhibited an area of inhibition zones of size 0.9 and 0.8 cm, respectively. The chromatogram for Rf1 showed five major peaks and several minor peaks. The retention time (Rt) of 3 major peaks (4.52, 50.99 and 59.56 min) were superimposable with the Rt of standards for gallic acid, cinnamic acid and flavone indicating the presence of above said phenolic compounds in detectable amounts. No characteristic peaks in the sample that corresponded with the retention time of the standards of vanillic acid, caffeic acid and 8-hydroxy quinoline were seen indicating their absence or presence in non detectable amounts. The major peak in the chromatograms of Rf1, Rf2 and Rf3 indicated that flavone is one of the most significant compounds in the leaf of D. metel. The above said phenolics may be responsible for the antifungal activity. Antifungal Table 6 Effect of purified phenolics from preparative TLC on the mycelial growth of Fusarium oxysporum f. sp. cubense. Eluted phenolics from TLC Rf value Color of the spot Inhibition zone (cm)* Rf1 Rf2 Rf3 Control (methanol only) 0.20 0.16 0.10 – Blue 0.90a 0.80b 0.70c Nil * Mean of five replications. Means followed by common letter are not significantly different at 5% level by DMRT. compounds were purified initially using TLC on silica gel from D. metel. Fraction of the TLC purified sample was test verified for antifungal activity. Fraction 1 was characterized through ESI-LC–MS. HPLC analysis denoted the presence of 24 various compounds in the fraction 1. Based on the molecular and fragmentation ions derived from ESI-LC–MS, three major compounds (two glycosides and one ester) such as 5,40 -dihydroxy, 7-O-glycosyl, 30 -methoxy flavone, 5,40 -dihydroxy, 7-O-pentosyl, 30 -methoxy flavone and triacontanol ester were identified in the fraction 1. 3.5.1. 5,40 -dihydroxy, 7-O-glycosyl, 30 -methoxy flavone The structure of this compound was given in Fig. 3A. The molecular ion of this compound showed a peak at m/z 594. In addition, it showed many fragmentation peaks. Loss of pentose ion (Arabinose?) from the molecular ion of the compound gave an ion at m/z 459 indicating the presence of pentose sugar (Arabinose?) in the original compound. A subsequent loss of glucose (179 Da) from the fragmentation ion at 459 gave an ion at m/z 297 indicating the presence of glucose in the original molecule. 3.5.2. 5,40 -dihydroxy, 7-O-pentosyl, 30 -methoxy flavone The structure of this compound was given in Fig. 3B. The molecular ion of this compound shows a peak at m/z 432. Loss of pentose ion (Arabinose?) from the molecular ion of the compound gave a peak at m/z 283 denoting the presence of a pentose (Arabinose?) in this compound. 3.5.3. Triacontanol ester This compound consists of an alcohol (triacontanol) and a 30 carbon fatty acid (tricontanoic acid). The structure of this compound was presented in Fig. 3C. The molecular ion of this compound shows a peak at m/z 874. Loss of 30 carbon fatty acid gave a peak at m/z 437, denoting the presence of tricontanoic acid. 4. Discussion Fig. 2. Native polyacrylamide gel electrophoresis (PAGE) analysis of defense enzymes in banana plants treated with bioagents. (A) peroxidase, (B) polyphenol oxidase. Nowadays botanical fungicides are gaining momentum in the management of plant pathogens. The mechanism of disease suppression by plant products and biocontrol agents have suggested that the active principles present in them may either act on pathogen directly or induce systemic resistance in host plants resulting in reduction of disease development (Paul and Sharma, 2002). Plant extracts are considered as an alternative source for chemicals in the management of soil borne pathogens. In the present study, 22 plant species were tested for their effectiveness against Foc. Among the plants screened, the aqueous leaf extract of D. metel (10%) completely inhibited the mycelial growth. Methanolic extract of D. metel was highly inhibitory to the mycelial growth of Foc compared to other solvents. The antifungal compounds present in this leaf extract may have prominent effect in inhibiting the mycelial growth of the pathogen (Kagale et al., 2004). The efficacy of PGPR against fungal, bacterial and viral diseases has been reported by various scientists (Kloepper et al., 2004; R. Akila et al. / Biological Control 57 (2011) 175–183 Harish et al., 2009a). The present study revealed that PGPR, Pf1 and TRC 54 were most effective in reducing the mycelial growth of Foc. These PGPR strains secrete antibiotics such as phenazine-1-carboxyclic acid, 2,4-diacetyl phloroglucinol, oomycin, pyoluteorin, pyrrolnitrin etc. which show antagonistic action against plant pathogens (Thomashow et al., 1997; Ayyadurai et al., 2006). Also they produce lytic enzymes like chitinase which can degrade the fungal cell wall (Radjacommare et al., 2004). Hence the reduction in mycelial growth observed in this study may be due to direct action of the enzymes and antibiotics produced by PGPR. Application of mixtures of botanical and biocontrol formulations Wanis + Pf1 + TRC and Damet + Pf1 + TRC 54 was very effective in reducing the Fusarium wilt incidence both under greenhouse and field conditions. Previous reports demonstrated that foliar application of methanolic leaf extract of D. metel significantly reduced the severity of rice sheath blight (33.3%) and bacterial blight disease (13.8%) when compared to control which recorded 71.6% and 33%, respectively under greenhouse conditions 181 (Kagale et al., 2004). Our results are in agreement with the finding of Rawal and Thakore (2003) who documented that the leaf extract (20%) of D. stramonium showed 75.04% inhibition of mycelial growth of F. solani which causes Fusarium rot of Sponge gourd. Also, mixtures of bacterial strains were found to be more effective in controlling many plant diseases when compared to a single strain as multiple mode of action may be involved (Kavino et al., 2007; Harish et al., 2008). In our study also similar effect with reduced disease incidence and high yield was noticed when mixtures of botanical and biocontrol formulations were used. In this experiment it was clear, the Wanis 20 EC + Pf1 + TRC 54 consortia resulted in an increase in plant growth parameters viz., plant height, pseudostem girth, leaf length and leaf breadth both under greenhouse and field conditions. Kishore and Pande (2005) reported that four sprays of D. metel leaf extract on groundnut was partially effective against foliar diseases (late leaf spot and rust) upto 95 DAS, in addition to an increase in the pod yield up to 91% over control. This indicates that some active principles in plant Fig. 3. Molecular structures of the compounds in D. metel (A) 5,40 -dihydroxy, 7-O-glycosyl, 30 -methoxy flavone, (B) 5,40 -dihydroxy, 7-O-pentosyl,30 -methoxy flavone and (C) triacontanol ester. 182 R. Akila et al. / Biological Control 57 (2011) 175–183 products are having growth promoting activity as well as antifungal property. Phytohormones produced by PGPR play a major role in growth promotion and many bacteria have the ability to produce auxins, gibberellins, cytokinins and ethylene (García de Salamone et al., 2001; Bottini et al., 2004). Some PGPR possesses ACC deaminase which lowers the ethylene level and thus indirectly promotes the growth of the plant (Saravanakumar et al., 2007). Recently Kavino and co-workers (2010) reported that CHA0 + chitin bio-formulation significantly increased the morphological characters and yield of banana as well as ratoon crops. Thus the increase in yield observed in the present study may be attributed to the combined growth promoting activity of the botanical and biocontrol formulations. Plants are endowed with various defense related genes connected with the induction of defense enzymes. Peroxidase is one of the defense enzymes which has been implicated in the last enzymatic step of lignin biosynthesis, that is, the oxidation of hydroxy cinnamyl alcohols into free radical intermediates, which subsequently are coupled into the lignin polymer (Gross, 1980). Furthermore, peroxidase itself inhibited the spore germination and mycelial growth of Pseudocercospora abelmoschi and Pseudocercospora cruenta (Joseph et al., 1998). In this work, plants treated with Wanis + Pf1 + TRC 54 recorded the highest peroxidase activity followed by Damet + Pf1 + TRC 54 and Pf1 + TRC 54. Kagale and co-workers (2004) reported that the application of methanolic leaf extracts (D. metel) induced the peroxidase activity in rice plants which were post inoculated with the sheath blight pathogen R. solani. Increase in phenolic compounds and peroxidase in banana were positively correlated with resistance to Fusarium wilt (Sariah et al., 2001). Marpago et al. (1994) indicated that the activity of peroxidase was at least five times higher in the roots and corm tissues of F. oxysporum resistant banana cultivar than the susceptible cultivar. The authors concluded that PO activity can be used as a parameter to discriminate between susceptible and tolerant clones of banana against Foc. Native gel electrophoresis revealed expression of more isoforms of PR proteins, peroxidase and chitinase in the banana plants challenged with mixtures of plant growth promoting endophytic bacteria and viruliferous aphids (Harish et al., 2009b). In our study, native PAGE analysis of peroxidase isozyme revealed that the isozyme PO1 was specifically observed only in the plants treated with Damet 50 EC + Pf1 + TRC 54 and Wanis 20 EC alone. Polyphenol oxidase is a copper containing enzyme, which oxidizes phenolics to highly toxic quinones and involved in the terminal oxidation of diseased plant tissues and is attributed for its role in disease resistance (Kosuge, 1969). The highest activity of PPO was observed in the roots of plants treated with Wanis 20 EC + Pf1 + TRC 54. PPO activity reached its maximum on 9th day after treatment and then it started declining. This result coincides with the finding of Saravanan et al. (2004) who documented that the PPO enzyme in root reached its maximum activity on 8th day after the application of Pf1. TLC purified phenolics of D. metel were further separated using RP-HPLC and identified by comparing with phenolic standards. The chromatograms revealed the presence of gallic acid, cinnamic acid, flavone, coumarin and syringic acid. Girijashankar and Thayumanavan (2005) identified the phenolics in the aqueous extract of L. inermis by RP-HPLC and added that the presence of phenolic compounds along with other phytochemical constituents resulted in the in vitro inhibition of mycelial growth of Rhizoctonia solani, Pythium aphanidermatum and Macrophomina phaseolina. Many plant phenolic compounds are known to be antimicrobial, function as precursors to structural polymers such as lignin, or serve as signal molecules (Nicholson and Hammerschmidt 1992; Stachel et al., 1986). Presence of phenolic acids such as gallic acid, syringic acid and cinnamic acid are one of the reasons responsible for the antifungal nature of the D. metel. Ahn et al. (2005) also reported the presence of gallic acid and methyl gallate in the galls on the nutgall sumac tree. Gallic acid exhibited strong antifungal activity against Magneporthe grisea. In the present study, two flavones (5,40 -dihydroxy, 7-O-glycosyl, 30 -methoxy flavone and 5,40 -dihydroxy, 7-Opentosyl, 30 -methoxy flavone) were found in the TLC purified fraction 1 by mass spectrometric analysis. The antifungal nature of flavones was already demonstrated. Cotoras et al. (2001) isolated two flavones from the resinous exudates of Pseudognaphalium spp. and reported that the flavone (5,7-dihydroxy-3,8-dimethoxy flavone) at 40 lg/ml concentration reduced the mycelial growth of B. cinerea by 32.1 per cent and another flavone (5,8-dihydroxy-3,6,7-trimethoxy flavone) reduced the hyphal growth by 14.9 per cent. Soil borne pathogens like Foc cannot be kept under control when we adopt a single management strategy. In the present study an integrated approach was carried out to manage this disease. Thus when we use compatible mixtures of botanical and biocontrol formulations, a significant reduction in the disease incidence besides growth promotion is achieved. 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