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The main objective of this study was to evaluate the effectiveness of crude chloroform extract of Piper betle L. (PbC) in controlling Fusarium wilt of tomato (Lycopersicon esculentum) caused by Fusarium oxysporum f. sp. lycopersici. It was observed that 1% (w/w) amendment of the PbC in soil was more efficient in reducing the Fusarium population in soil than carbendazim and the combined amendment of carbendazim and PbC. Fusarium wilt control studies were carried out in a greenhouse. Variation in different parameters like shoot growth, root growth and mean fresh weights of tomato seedlings in all the treatments were recorded. Accumulation of total phenolics was also studied from the root tissues of tomato. Higher accumulation of total phenolics was observed in the Fusarium-infested plants as compared to that of healthy control and PbC-treated plants. Moreover, it was observed that the extract could reduce the symptoms and disease development. Electron microscopy studies were also done to observe the Fusarium infestation in the vascular bundles and to show the accumulation of total phenolics in the vacuoles of root tissue.[PUBLICATION ABSTRACT]
World J Microbiol Biotechnol (2011) 27:25832589 DOI 10.1007/s11274-011-0730-6
ORIGINAL PAPER
Control of Fusarium wilt of tomato caused by Fusarium oxysporum f. sp. lycopersici using leaf extractof Piper betle L.: a preliminary study
Irom Manoj Singha Yelena Kakoty Bala Gopalan Unni
Mohan Chandra Kalita Jayshree Das Ashok Naglot
Sawlang Borsingh Wann Lokendra Singh
Received: 7 December 2010 / Accepted: 21 March 2011 / Published online: 1 April 2011 Springer Science+Business Media B.V. 2011
Abstract The main objective of this study was to evaluate the effectiveness of crude chloroform extract of Piper betle L. (PbC) in controlling Fusarium wilt of tomato (Lycopersicon esculentum) caused by Fusarium oxysporumf. sp. lycopersici. It was observed that 1% (w/w) amendment of the PbC in soil was more efcient in reducing the Fusarium population in soil than carbendazim and the combined amendment of carbendazim and PbC. Fusarium wilt control studies were carried out in a greenhouse. Variation in different parameters like shoot growth, root growth and mean fresh weights of tomato seedlings in all the treatments were recorded. Accumulation of total phenolics was also studied from the root tissues of tomato. Higher accumulation of total phenolics was observed in the Fusarium-infested plants as compared to that of healthy control and PbC-treated plants. Moreover, it was observed that the extract could reduce the symptoms and disease development. Electron microscopy studies were also done to observe the Fusarium infestation in the vascular bundles and to show the accumulation of total phenolics in the vacuoles of root tissue.
Keywords Fusarium wilt Piper betle L. Tomato
Introduction
Fusarium species causes a huge range of diseases on an extraordinary range of host plants. The fungus can be soilborne, airborne or carried in plant residue and can be recovered from any part of the plant from the deepest root to the highest ower (Booth 1971; Summeral et al. 2003). Fusarium wilt of tomato (Lycopersicon esculentum) caused by Fusarium oxysporum f. sp. lycopersici is a disease that causes serious economic loss (Agrios 2005). The fungus causes vascular wilts by infecting plants through the roots and growing internally through the cortex to the stele (Bowers and Locke 2000). Control of wilt diseases depends mainly on fungicides (Minton 1986; DeVay et al. 1988). Several fungicides have been used for control of different plant pathogens including fusaria (Liggit et al. 1997; Diehl and Fehrmann 1999) and the number of effective fungicides with negligible effect on the environment is rare. Fungicides are expensive, can cause environmental pollution and may cause the selection of pathogen resistance (Lumsden and Locke 1989). Alternative methods of controlling the disease have been studied with emphasis on novel compounds derived from plant sources (Garibaldi et al. 1990; Alabouvette 1999). Plant extracts and plant essential oils have been reported to be effective antimicrobials against food and grain storage fungi, foliar pathogens and soilborne pathogens (Bowers and Locke 2000). Many plants and their products have been reported to possess pest control properties. These are good alternatives to chemical pesticides, as they are readily biodegradable in nature. In our previous study, we observed that chloroform extract of Piper betle L. (PbC) was found to be effective in controlling phytopathogenic fungi of economic importance in vitro (Singha et al. 2010). So, an attempt was made to study the efcacy of the Piper extract to control
I. M. Singha J. Das A. Naglot L. Singh
Biotechnology Division, Defence Research Laboratory (DRDO), Tezpur 784 001, Assam, India
Y. Kakoty B. G. Unni (&) S. B. Wann
Biotechnology Division, North-East Institute of Science and Technology (CSIR), Jorhat 785 006, Assam, Indiae-mail: [email protected]; [email protected]
M. C. KalitaDepartment of Biotechnology, Gauhati University, Guwahati 781 014, Assam, India
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the Fusarium wilt of tomato in vivo. In this study, we report the effectiveness of crude chloroform extract ofP. betle L. (PbC) in reducing populations of F. oxysporum in soil and controlling wilt development in tomato plants.
Materials and methods
Inoculum preparation
Fusarium oxysporum F1 (Acc. No. HM802271), F2 (Acc. No. HM802272) and F5 (Acc. HQ332534) were used in this study. For inoculum preparation, the fungus was cultured in potato dextrose broth (PDB; Himedia, Mumbai) enriched with yeast extract (Himedia, Mumbai) at 10 g l-1
in shake culture (150 rev min-1), for 10 days at 28 2C. The broth culture was ltered through 4 layers of muslin cloth, centrifuged at 6,000g for 10 min and washed with sterile water. The test inoculum was adjusted to 2.1 9 106 spores per ml using a haemocytometer. Inoculum consisted mainly of microconidia.
Plant material
Leaves of P. betle L. were collected and shade dried. It was grounded into ne powder and solvent extracted using petroleum ether for 48 h. It was then ltered using lter paper no. 1 (Whatman, Mumbai) and the residue thus obtained was dried completely. It was again extracted using chloroform (Merck, Mumbai) for 48 h and ltered. The ltrate was dried in a rotary evaporator (Heidolph, Germany). The dried crude extract was diluted in appropriate concentrations of dimethylsulfoxide (Himedia, Mumbai). Qualitative analysis of the chloroform extract of P. betle L. was carried out using standard methods. Seeds of the Fusarium wilt susceptible tomato plant (Pusa Ruby) were surface disinfected with 1% sodium hypochlorite (Himedia, Mumbai) for 2 min and rinsed three times with sterile distilled water prior to sowing.
Soil infestation and treatment
Potting mixtures (pH 6.8) which is amended with KNO3(0.3 g l-1), KH2PO4 (0.18 g l-1) and NH4NO3 (0.3 g l-1) was used. Sterile potting mixture was infested with spores of
Fusarium oxysporum. It was incubated at 28 2C in sterile plastic bags for 57 days or till the fungal population reached 106 spores per gram of the soil. Uninfested soil was used as the negative control. For control of Fusarium wilt, the fungus-infested soil was treated with different concentrations of chloroform extract of P. betle L. (PbC) and carbendazim (BASF, Mumbai) under standard conditions. The experiment was carried out in a greenhouse.
Control of Fusarium wilt
For control of Fusarium wilt in the greenhouse, six-week-old tomato seedlings were planted in plastic bags (12 cm diameter; 1 l capacity) containing soil treatments. The treatments included: (a) healthy control (no fungus);(b) fungal infested control; (c) infested soil amended with DMSO; (d) infested soil amended with 0.1, 0.5, 1, 2 and 5% (w/w) PbC; (e) infested soil amended with 0.1, 0.5, 1, 2, and 5% (w/w) carbendazim and (f) infested soil amended with 0.1, 0.5, 1, 2 and 5% (w/w) combined PbC and carbendazim (1:1). Three different Fusarium isolates: highly virulent F1, virulent F2 and avirulent F5 were also included in the study to assess the activity of the plant extract on the Fusarium soil population and disease incidence. Soil samples of respective treatments were collected and the populations of the fungus were assessed in vitro using the soil dilution method (Aneja 2005). The soil dilutions were plated in pentachloronitrobenzene (PCNB; SigmaAldrich, Germany) agar medium (Nash and Snyder 1962), incubated at 28 2C for 35 days and appearance of Fusarium colonies was noted. Variation in shoot growth, root growth, fresh weight of seedlings and variation in symptom developments were observed in all the treatments for a period of 28 days after planting. The disease severity were recorded on a 03 visual scale, in which 0 = no symptoms; 1 = light yellowing of leaves; 2 = moderate or severe yellowing of leaves with or without wilting, stunting, severe rot on taproot and secondary roots, crown rot with or without hypocotyls rot and vascular discolouration in the stem; and 3 = dead seedlings (Vakalounakis and Fragkiadakis 1999). Disease incidence percentage was determined according to Song et al. (2004). The experiments were carried out in a greenhouse (2228C, 7080%
relative humidity and 12 h photoperiod). Each treatment consisted of 25 replicates and was repeated twice.
Determination of accumulation of phenolics in root tissue
The total phenolics content in tomato roots was determined using the Folin-Ciocalteu method (Policegoudra et al. 2007) with slight modications. Briey 100 ll of the methanolic root extracts of all treatments were mixed with 2 ml of 2% aqueous sodium carbonate solution (Himedia, Mumbai). After 5 min 100 ll of 50% Folin-Ciocalteu phenol reagent (SRL, Mumbai) was added to the mixture. Incubation was done at room temperature for 30 min and the absorbance was recorded at 750 nm against a blank using a spectrophotometer (Thermo Spectronic UV1, USA). Calibration curve was prepared using gallic acid (SRL, Mumbai) as the standard reference phenolic compound and thus total phenolics content was calculated. All
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determinations were carried out in triplicates. The total content of phenolics in the root extract was calculated as lg g-1 fresh weight (FW) root tissue.
Electron microscopy analysis of tomato root tissue
Scanning electron microscopy (SEM) of root tissue was done using a JEOL JSM 6360 SEM operating at 20 kV. Transmission electron microscopy (TEM) was done using a JEOL JSM 100-CX TEM operating at 60 kV. These studies were carried out at Sophisticated Analytical Instrument Centre (SAIF), NEHU, Shillong, India.
Statistical analysis
Statistical calculations were carried out using one way analysis of variance (ANOVA) and the signicances of the differences between means were calculated using Duncans multiple range test under a signicance level of P \ 0.05.
Results
Qualitative phytochemical test
PbC showed the presence of saponins, phlobatanins, avonoids, phytosterols, phenols, tannins and terpenoids.
Fusarium soil population assay
There was no Fusarium colony in the uninfested control (healthy) while in the Fusarium-infested control and DMSO control. There was almost the same number of Fusarium colonies. It was observed that PbC was more efcient than carbendazim and combined activity of carbendazim and PbC in reduction of the fungal population in all the treatments (Fig. 1). 1% PbC amendment in soil could successfully inhibit the Fusarium soil population. It was observed there was no signicant difference in the inhibition of Fusarium population on the three different isolates: highly virulent Fusarium F1, virulent Fusarium F2 and avirulent Fusarium F5 by PbC (Table 1).
Effect of soil amendment with PbC on Fusarium
The growth of plants was stunted in fungus-infested soil as compared to the healthy normal control. Plants grown in Fusarium-infested soil amended with 1% (w/w) PbC attained greater shoot height (Fig. 2a) as compared to the diseased plants. It may be compared with that of the healthy control after 21 days of planting. The increase in the shoot height was statistically signicant (P \ 0.05) in all treatments on all days of observation. Seedlings planted
in Fusarium-infested soil without PbC amendment had decreased root growth. The root growth of seedlings planted in Fusarium-infested soil amended with 1% (w/w) PbC attained comparable growth with that of the healthy control after 28 days of planting. The results were statistically signicant (P \ 0.05) on all days of observation (Fig. 2b). Plants from Fusarium-infested soil amended with 2% (w/w) PbC attained comparable fresh weights with that of healthy control than plants from Fusarium-infested soil or the PbC treatment at 0.5% (w/w) PbC. The results were statistically signicant (P \ 0.05) (Fig. 2c). The PbC amendment in Fusarium-infested soil decreased the wilting symptoms and disease developments. The suppression of disease and symptom development for all treatments was signicant on all days of observation. 1% (w/w) PbC amendment in soil gradually reduced the Fusarium population in soil. Only a negligible 10% disease incidence was observed after 28 days as compared to 65% disease incidence at 15 days of treatment (Fig. 2d). The results were statistically signicant (P \ 0.05) on all days of observation. It was also observed that delayed amendment of PbC in soil after 15 days of Fusarium F1 infestation could reduce the disease incidence form 88 to 44% only.
Determination of accumulation phenolics in root tissue
The fungus-infested seedlings had an increased content of total phenolics in their root tissues. The content increased to a maximum at 21 days in all treatments and then got gradually reduced except the fungal-infested seedlings. It was observed that there was a decline in the accumulation of total phenolics in the healthy control and the PbC amendments after 21 days of observation. But the Fusarium-infested treatments showed more accumulation of total
Fig. 1 Fusarium soil population assay in treatment with different concentrations of chloroform extract of P. betle L., carbendazim and combined treatment of chloroform extract of P. betle L. and carbendazim. Values with different letters are signicantly different at P \ 0.05
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Table 1 Activity of chloroform extract of Piper betle L. on the population density and disease incidence of different Fusarium oxysporum isolates after 28 days of observation
Fusarium isolates Population density (104 g-1 soil) Disease incidence
Control 1% PbC amended soil Control 1% PbC amended soil
F1 (extremely virulent) 107 3.0a 4.6 2.1a 88.8 2.9a 11 0a
F2 (virulent) 108 4.6a 3 1.8a 36.0 8.0b 4.4 2.7b
F5 (avirulent) 101 2.0a 7 2.3a 7.2 3.4c 0 0c
PbC chloroform extract of Piper betle L.; Values showed by different letters for each line is signicantly different at P \ 0.05. Values are given as mean values standard deviation
Fig. 2 Effect of tomato plants grown in soil amended with chloroform extract of Piper betle L. for control of Fusarium wilt. a variation in shoot growth; b variation in root growth; c variation in weight;
d variation in disease incidence of tomato seedlings. Values showed by different letters for each line is signicantly different at P \ 0.05
phenolics even after 21 days of treatment, and reached the maximum at 28 days of observation (Fig. 3). The results were statistically signicant (P \ 0.05).
Electron microscopy analysis of tomato root tissue
It was evident from the scanning electron microscopic observations that Fusarium blocked the vascular bundles giving much insight into the fungal infestation in the Fusarium-infested tomato plants (Fig. 4b), whereas the healthy (Fig. 4a) and the PbC-amended seedlings showed clear vascular bundles (Fig. 4c). From the studies by transmission electron microscopy, a higher level of accumulation of total phenolics was observed in the vacuole of the Fusarium-infested tomato root tissue (Fig. 5b). The
healthy control (Fig. 5a) and the 1% (w/w) PbC amendment had a normal accumulation of total phenolics. Also the healthy control was observed to have a cell wall with good lignications (Fig. 5c) when compared with the cell wall of the Fusarium-infested root tissue (Fig. 5d).
Discussion
From our investigation, it was observed that there was a reduction in symptom development and as well as in Fusarium population after 28 days of observation in the tomato plants grown in soil treated with 1% (w/w) chloroform extract of P. betle L. (PbC). There was a signicant increase in the shoot growth, root growth and mean fresh
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weight of tomato plants as compared with the fungal control. This increase in all these parameters can be well compared with that of the healthy control on all days under observation. In the case of plants grown in soil amended with 2% (w/w) PbC, it was observed to be better than that of healthy control. It is a common phenomenon that Fusarium wilt generally attacks tomato plants during the growth period of 60 days, when owering starts. The pathogen enters through the roots of the plant and proliferates in the vascular tissues leading to breakdown of the water economy of the infected plants (Agrios 2005). Singha et al. (2010) observed that the chloroform extract of P. betle L. was thermally stable at room temperature even up to 120 days of storage and more, with similar antifungal properties as the fresh extract. It was also observed that chloroform extract of P. betle L. was best effective when
Fig. 3 Variation in accumulation of total phenolics in root tissue of tomato seedlings grown in soil with different amendments of chloroform extract of Piper betle L. Values showed by different letters for each line are signicantly different at P \ 0.05
Fig. 4 Scanning electron microscopic slides showing a vascular bundles (VB) of tomato plants in healthy control; b Fusarium infestation (FM) in vascular bundles (VB) of tomato plants in fungal
control and c clear vascular bundles (VB) showing no Fusarium infestation in the plants grown in soil treated with 1% (w/w) chloroform extract of P. betle L. (PbC)
Fig. 5 Accumulation of total phenolics (shown by arrow) in vacuoles (V) of a healthy root tissue, Bar 0.37 lm;
b Fusarium-infested root tissue, Bar 0.37 lm; c cell wall (CW)
in healthy control, Bar 1.25 lm and d cell wall in Fusarium-infested root tissue, Bar1.25 lm
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stored at 4C. Terpenoids were observed to be present in PbC amongst other phytochemicals viz. saponins, phlobatanins, avonoids, phytosterols, phenols and tannins. Since delayed amendment of PbC in soil after 15 days of Fusarium F1 infestation could reduce the disease incidence from 88 to 44%, it can be speculated that some unknown volatile aromatic compounds present in the form of terpenoids from PbC may help in conferring disease resistance inside the vascular tissues of tomato plants. Minerdi et al. (2009) demonstrated that small volatile organic compounds (VOCs) sesquiterpenes, mainly caryophyllene, emitted from the wild type strain negatively inuence the mycelial growth of different formae speciales of F. oxysporum.
High content of phenolics may be an essential component for defense against various pathogens that constantly challenge the plant body. Phenolics from various plant sources and their contribution to antimicrobial and other biological responses are well documented (Benner 1993; Bennet and Wallsgrove 1994). It was observed that there was a decline in the accumulation of total phenolics in the healthy control and the PbC amendments after 21 days of observation. But the Fusarium-infested treatments showed more accumulation of total phenolics even after 21 days of treatment, and reached the maximum at 28 days of observation. Decrease in the phenolics may be attributed to strengthening of the plant cell walls by polymerization into lignans and lignins (Randhir and Shetty 2005). Phenolic compounds produced by plants are formed through phenylpropanoid metabolism. Since free phenolics can be cytotoxic in the cytoplasm, plants sequester these compounds in the vacuole or deposit them in the cell wall. Once the phenolic acids reach the cell wall, they may be either ester- or ether- linked to the cell wall polysaccha-rides or hemicelluloses, or polymerized into lignin (Lewis and Yamamoto 1990). The role of lignins and related polymers has been closely correlated with the defense responses of several plants (Vance et al. 1980). Phenols occur constitutively, whereas others are formed in response to pathogen ingress and associated as part of an active defense response in the host (Nicholson and Hammerschmidt 1992). They are known to confer resistance either directly or indirectly through activation of postinfection responses in the hosts (De Vecchi and Matta 1989). Post-infection activation of phenol metabolism in xylem vessels of tomato has been demonstrated by Matta et al. (1988). The increase in the synthesis and accumulation of total phenolics in the plants grown in Fusarium-infested soil may be due to resistance and overcome the stress created by the invading Fusarium. It further suggests that only an increase in the synthesis and accumulation of total phenolic compounds in the plant body is insufcient to resist and check the entry of Fusarium. So proper sequestration of these phenolic compounds in the form of lignications in
the cell wall is necessary for the plant defense mechanism to confer resistance and overcome the entry of pathogens.
Generally biocontrol and plant secondary metabolites have been known to stimulate the production of phenolics and other compounds that constitutively help the plants in disease resistance. Amendment of PbC in soil reduced the Fusarium soil population. The extract effectively checks the Fusarium population and its growth in soil and gradually reduces disease symptoms and renders effective growth to tomato plants. However, little is known about the ability of PbC to stimulate higher levels of defense enzymes and host defense related compounds. The characterization of the secondary metabolite from the crude chloroform extract of Piper betle L. was determined by other workers in their study (Parmar et al. 1998), but further investigations are required. Thus, isolation and identication of active compounds associated with antifungal activity from chloroform extract of Piper betle L. may serve as a promising alternative to chemical fungicides and in management of Fusarium wilt of tomato and other soil borne pathogens.
Acknowledgments We thank Dr. P. G. Rao, Director, NEIST, Jorhat and Dr. R. B. Srivastava, former Directorate of Life Sciences, DRDO, New Delhi for constant support and encouragement. We thank Dr. P. Chattopadhyay and Dr. R. S. Policegoudra for valuable guidance. Dr. B. G. Unni gratefully acknowledges Defence Research and Development Organization (DRDO), Govt. of India for nancial support. I. M. Singha acknowledges DRDO for the SRF fellowship.
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