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Natural Product Research: Formerly Natural Product Letters Publication details, including instructions for authors and subscription information: http://www.tandfonline.com/loi/gnpl20
Nematicidal activity of microbial pigment from Serratia marcescens a
a
a
Suryawanshi Rahul , Patil Chandrashekhar , Borase Hemant , b
a
ac
Narkhede Chandrakant , Shinde Laxmikant & Patil Satish a
School of Life Sciences, North Maharashtra University, Jalgaon, Maharashtra 425001, India b
Applied Parasitology Research Lab, Department of Zoology, J. E. S. College Jalna, Aurangabad, Maharashtra 431203, India c
North Maharashtra Microbial Culture Collection Centre, North Maharashtra University, Maharashtra, India Published online: 04 Apr 2014.
To cite this article: Suryawanshi Rahul, Patil Chandrashekhar, Borase Hemant, Narkhede Chandrakant, Shinde Laxmikant & Patil Satish (2014) Nematicidal activity of microbial pigment from Serratia marcescens, Natural Product Research: Formerly Natural Product Letters, 28:17, 1399-1404, DOI: 10.1080/14786419.2014.904310 To link to this article: http://dx.doi.org/10.1080/14786419.2014.904310
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Natural Product Research, 2014 Vol. 28, No. 17, 1399–1404, http://dx.doi.org/10.1080/14786419.2014.904310
SHORT COMMUNICATION Nematicidal activity of microbial pigment from Serratia marcescens Suryawanshi Rahula, Patil Chandrashekhara, Borase Hemanta, Narkhede Chandrakanta, Shinde Laxmikantb and Patil Satishac*
Downloaded by [University of Newcastle (Australia)] at 10:11 03 September 2014
a
School of Life Sciences, North Maharashtra University, Jalgaon, Maharashtra 425001, India; Applied Parasitology Research Lab, Department of Zoology, J. E. S. College Jalna, Aurangabad, Maharashtra 431203, India; cNorth Maharashtra Microbial Culture Collection Centre, North Maharashtra University, Maharashtra, India b
(Received 6 January 2014; final version received 11 March 2014) Ineffectiveness of available nematicides and the high damage caused by plant-parasitic nematodes result in the urgent need to find some natural remedy for their control. Bioactivity of the pigment extracted from Serratia marcescens was screened for controlling nematodes at their juvenile stage. Test pigment was found effective against juvenile stages of Radopholus similis and Meloidogyne javanica at low concentrations (LC50 values, 83 and 79 mg/mL, respectively) as compared with positive control of copper sulphate (LC50 values, 380 and 280 mg/mL, respectively). The pigment also exhibited inhibition on nematode egg-hatching ability. Characterisation of extracted pigment with TLC, FTIR, HPLC, HPTLC and spectroscopic analysis confirmed the presence of prodigiosin as a bioactive metabolite. Considering the sensory mechanism of pathogen recognition by nematodes, the use of microbial secondary metabolites can be effective for nematode control rather than using whole organism. Keywords: pigment; nematode; Radopholus similis; Meloidogyne javanica; Serratia
1. Introduction Plant-parasitic nematodes are worm-shaped microscopic animals. Nematodes may cause negligible injury or even total destruction of plant material. Mode of damage to plants by nematodes can be different, it may be reducing or modifying root mass, may act as predisposing agents; for example spread of bacterial canker, they make plants more susceptible to certain fungal pathogens such as Verticillium wilt (Frank 1992) and can cause hormonal imbalance in the root. In addition, nematodes act as vectors for viral infections in plants (Brown et al. 1995), for example both tomato ringspot virus and tobacco ringspot virus are transmitted from infectious plant to normal plant by a common species of dagger nematodes (Xiphinema spp.). Chemical nematicides such as 1,2-dichloropropene, 1,2-dichloropropane, ethylene dibromide, methyl bromide, chloropicrin, metam sodium, dazomet, methyl isothiocyanate, carbamates and organophosphates have been proved to be ecologically unfavourable due to various reasons (Schneider et al. 2003). Environmental problems associated with the use of chemical nematicides have led to a sense of urgency to investigate alternative methods for nematode management (Kerry 2000). Micro-organisms can inhibit plant-parasitic nematodes by use of secondary metabolites, enzymes and toxins. Their manifestation may include suppression of nematode reproduction, egg hatching and juvenile survival, or direct killing of nematodes (Siddiqui & Mahmood 1999).
*Corresponding author. Email: [email protected] q 2014 Taylor & Francis
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Bacteria exhibiting nematicidal effects include members of genera Rhizobium (Neipp & Becker 1993), Enterobacter (Duponnois et al. 1999), Actinomycetes (Yavuzaslanoglu et al. 2011), Burkholderia (Meyer et al. 2001), Serratia (El-Sherif et al. 1999), Chromobacterium (Don et al. 1997), Azotobacter, Bacillus, Clostridium, Corynebacterium and Methylobacterium (Prabhu et al. 2009). Rhizobacteria are the only group of micro-organisms in which biological nematicides have been reported, besides the nematophagous fungi and Actinomycetes (Baoyu et al. 2007). Most thoroughly studied organism for nematicidal activity is Bacillus subtilis (Siddiqui & Shaukat 2002). Bio Yield (Gustafson LLC, Plano, TX, USA) and BioNem are few commercially available products based on Bacillus nematicidal activity (Giannakou & Prophetou 2004). Few Cry proteins from Bacillus thuringiensis are also known for nematicidal potential. Serratia marcescens, a Gram-negative bacterium, has been reported to produce cellassociated red-coloured pigment called prodigiosin (Carbonell et al. 2000). This deeply studied pigment exhibits inhibitory activity against Gram-positive micro-organisms, antifungal (Chandni et al. 2012), antiprotozoal (Genes et al. 2011), antimalarial, immunosuppressive (Tsuji et al. 1990), anticancer (Diaz et al. 2001) and insecticidal (Chandrashekhar et al. 2011) activities. These properties make prodigiosin a powerful research tool for screening against a range of plant and animal pathogens. S. marcescens as a whole organism has also been reported as a pathogenic strain for plant-parasitic nematodes. Considering the antibiotic potential of prodigiosin, it was screened against the juveniles of plant-parasitic nematodes associated with banana and brinjal. Crop losses caused by nematodes to these plants are very high. Worldwide, an average annual yield loss was estimated to be about 20% (Sesser & Freakman 1987). Toxicity studies of prodigiosin on agriculturally important micro-organisms (AIMs) have also been conducted to ensure eco safety aspects of pigment in actual field conditions. The pigment was characterised using different techniques. 2. Results and discussion After incubation of Serratia culture, the pigment was extracted from the biomass with acidified methanol. The crude extract was evaporated till complete drying. Furthermore, the dried pigment was purified by TLC and then quantified on dry weight basis. A total of 50 mg pigment was produced. In TLC analysis, the extract displayed an Rf value (0.87) similar to that of standard prodigiosin (Sigma-Aldrich, St. Louis, MO, USA) with butanol:hexane (2:1) as a solvent system (Figure S1). Spectroscopic studies indicated maximum absorbance at 535 nm (Figure S2), which is in accordance with results for prodigiosin from S. marcescens. The HPLC and HPTLC analyses revealed single peak at 536 and 535 nm, respectively, with a retention time of 13.4 min in HPLC (Figure S3). In FTIR analysis (Figure S4), red pigment from S. marcescens revealed strong absorption at 3516 per cm (NZH) and 2997 per cm (aromatic CZH), 1668 per cm (aromatic CvC) which suggests the presence of respective bands; prodigiosin exhibits similar absorptions in CHCl3 at 1630, 1602 per cm. Fingerprint region of the same displayed distinctive intensity band at 1668 per cm (aromatic Cv0), 1039, 1018 (CZO, CZN) carboxylic groups, 953, 899, 703 per cm (CZH) alkanes, 1313 (nitro compounds), 1434, 1410 (CZH) alkenes. Transmission pattern of the pigment was similar for the functional groups present in prodigiosin. Characterisation studies such as TLC, FTIR, HPLC, HPTLC and spectroscopic analysis confirmed the presence of prodigiosin in the sample. These results are in accordance with Song et al. (2006). Purified pigment was evaluated for nematicidal activity against plant-parasitic nematodes Radopholus similis and Meloidogyne javanica. In larvicidal bioassay, the pigment exhibited highly toxic action against juveniles of R. similis and M. javanica with LC50 values of 83, 79 mg/ mL and LC90 values of 272, 182 mg/mL, respectively. Copper sulphate was used as a positive
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control; it displayed LC50 values of 380, 280 mg/mL and LC90 values of 1810, 1040 mg/mL against R. similis and M. javanica, respectively (Figure 1 and Table 1). Same concentrations of pigment when tested for nematode egg hatching exhibited complete inhibition of the hatching ability of nematode eggs, and no juveniles were observed to be coming out of the eggs till four weeks for all tested concentrations (data not shown). Larvicidal assay demonstrated that the LC50 values presented by the pigment are significantly low when compared not only against positive control (copper sulphate) (Table 1) but also against synthetic pesticides such as chlorpyrifos, carbosulfan and deltamethrin, which presents LC50 values of 19.4, 25.3, and . 40 mg/mL, respectively, against Meloidogyne spp. (Wiratno et al. 2009). The concentration- and time-dependent study of prodigiosin and copper sulphate revealed that the prodigiosin possessed high nematicidal potential than copper sulphate at very low concentration and in a very short time (Figure 1). Our observation supports the pigment of S. marcescens as a stronger nematicidal candidature compared with copper sulphate and synthetic pesticides. Toxicity studies of prodigiosin against AIMs such as Bradyrhizobium japonicum, Azotobacter vinelandii and Trichoderma viride indicated normal growth of these AIMs in the presence of high prodigiosin concentration (500 mg/mL). Our observations suggest that the use of prodigiosin even at high concentrations is safe for agriculturally important flora of the soil. Insecticidal properties of S. marcescens are well known including the genera Anomala, Costelytra and Phyllophaga (Nunez et al. 2008); prodigiosin has been demonstrated to decrease egg-hatching ability in budworm Lepidoptera. It has also been reported to inhibit different stages of mosquito larvae (Aedes aegypti and Anopheles stephensi) (Chandrashekhar et al. 2011). Serratia can be used to control the diamondback moth, Plutella xylostella (Jeong et al. 2010). In another study, S. marcescens was found to reduce the development of root-knot nematode Meloidogyne incognita by high percentage reduction in the number of the juveniles in soil ranging from 77.2% to 84.4% in situ (Zeinat et al. 2009). S. marcescens was also used in
Figure 1. [A] Percent survival of juveniles (a) R. similis and (b) M. javanica against pigment, from Serratia with respect to time of incubation. [B] Percent survival of juveniles (c) R. similis and (d) M. javanica against positive control, from Serratia with respect to time of incubation.
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Table 1. Nematicidal activity of pigment prodigiosin from S. marcescens. Nematode
Test product
LC50 ^ SD (mg/mL)
LC90 ^ SD (mg/mL)
R. similis
Prodigiosin Positive control Prodigiosin Positive control
83 (^0.6) 380 (^8) 79 (^0.5) 280 (^4)
272 (^2.5) 1810 (^15) 182 (^7) 1040 (^10)
M. javanica
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Notes: Positive control, copper sulphate; SD, standard deviation; LC50, lethal concentration that kills 50% of juveniles; LC90, lethal concentration that kills 90% of juveniles. Level of significance P , 0.05.
agricultural field for the suppression of M. incognita to increase the yield of sunflower (Ali 1996). An enzyme, chitinase, from Serratia spp. is also reported to cause premature egg hatching of Meloidogyne helpa (Mercer et al. 1992). The effect of Serratia on nematodes appears to be strain specific as some researchers have reported symbiotic association of Serratia nematodiphila with nematodes such as Heterorhabditidoides chongmingensis. Several entomopathogenic nematodes such as Oscheius chongmingensis, Oscheius carolinensis and Caenorhabditis briggsae employ insect pathogenic bacteria such as Serratia to kill insects (Abebe et al. 2010; Ye et al. 2012; Torres et al. 2011). Though reports are available for the direct use of Serratia spp., as a biocontrol agent, its use may be limited since this approach could be pathogenic to animals as well (Jeong et al. 2010). Sensory mechanism for pathogen recognition has been reported in nematodes such as Caenorhabditis elegans. They are able to detect the pathogenic bacterium such as S. marcescens and can avoid the organism from entering inside the body, based on the production ability of the cyclic lipodepsipentapeptide serrawettin W2 (Elizabeth et al. 2007). Concerning this mechanism, the use of secondary metabolites rather than bacterial cells could be an effective way to control the growth of plant-parasitic nematodes. Prodigiosin from Serratia spp. possesses proton-sequestering ability (Roser et al. 2007), which affects intracellular pH gradient. Prodigiosin also affects the mitochondria by exerting an uncoupling effect on the electron chain transport of protons to mitochondrial ATP synthase; it causes reduction in ATP production without decreasing the oxygen consumption. Few other studies have also reported prodigiosin as a proton-pump inhibitor (Sato et al. 1998; Matsuya et al. 2000). Action of prodigiosin on the parasite Trypanosoma cruzi is found to be exerted by structural and functional disruptions of mitochondria, which might escort the parasites to an apoptotic-like cell death process (Genes et al. 2011). 3. Conclusion The use of secondary metabolites can be an effective way for nematode inhibition rather than the use of whole organism. Low concentrations of tripyrrol pigment from S. marcescens have been found to inhibit the juvenile stage of nematodes (R. similis and M. javanica); the LC50 value of the pigment is 5- to 10-fold less than other pesticides. The non-toxicity of prodigiosin against agriculturally important flora suggests the eco safety of the pigment. In order to explore the nematicidal potential of prodigiosin, field studies are required to confirm the infield activity. Specific reason for nematicidal action of prodigiosin merits further research. Supplementary material Experimental details relating to this article are available online, alongside Figures S1– S5.
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Acknowledgements We are thankful to Rajiv Gandhi Science and Technology Commission, Mumbai, Government of Maharashtra, India for financial assistance. We are also thankful to North Maharashtra Microbial Culture Collection Centre for providing microbial cultures.
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Roser F, Ricardo PT, Pepita GB, Vanessa SC, Pol GX, Santiago A. 2007. Mechanism of prodigiosin cytotoxicity in human neuroblastoma cell lines. Eur J Pharmacol. 572:111– 119. Sasser JN, Freckman DW. 1987. A world perspective on nematology: the role of resistance of banana cultivars to the nematodes Radopholus similis (Cobb) Thorne and Meloidogyne incognita Chitwood. Philipp Agric. 68:335–349. Sato T, Konno H, Tanaka Y, Kataoka T, Nagai K, Wasserman HH, Ohkuma S. 1998. Prodigiosins as a new group of Hþ/ Cl2 symporters that uncouple proton translocators. J Biol Chem. 273:21455–21462. Schneider SM, Rosskopf EN, Leesch JG, Chellemi DO, Bull CT, Mazzola M. 2003. Research on alternatives to methyl bromide: pre-plant and post-harvest. Pest Manag Sci. 59:814–826. Siddiqui IA, Shaukat SS. 2002. Rhizobacteria-mediated induction of systemic resistance (ISR) in tomato against Meloidogyne javanica. J Phytopathol Phytopathol Z. 150:469– 473. Siddiqui ZA, Mahmood I. 1999. Role of bacteria in the management of plant parasitic nematodes: a review. Bioresource Technol. 69:167–179. Song MJ, Bae J, Lee DS, Kim CH, Kim JS, Kim SW, Hong SI. 2006. Purification and characterization of prodigiosin produced by integrated bioreactor from Serratia sp. KH-95. J Biosci Bioeng. 101:157–161. Torres-Barragan A, Suazo A, Buhler WG, Cardoza YJ. 2011. Studies on the entomopathogenicity and bacterial associates of the nematode Oscheius carolinensis. Biol Control. 59:123–129. Tsuji RF, Yamamoto M, Nakamura A, Kataoka T, Magae J, Nagai K, Yamasaki M. 1990. Selective immunosuppression of prodigiosin 25-C and FK506 in the murine immune system. J Antibiot. 43:1293 –1301. Wiratno D, Taniwiryono H, Van DB, Riksen JAG, Rietjens IMCM, Djiwanti SR, Kammenga JE, Murk AJ. 2009. Nematicidal activity of plant extracts against the root-knot nematode, Meloidogyne incognita. Open Nat Prod J. 2:77–85. Yavuzaslanoglu E, Yamac M, Nicol JM. 2011. Influence of actinomycete isolates on cereal cyst nematode Heterodera filipjevi juvenile motility. Nematol Mediterr. 39:41–45. Ye WM, Torres BA, Cardoza YJ. 2012. Oscheius carolinensis n. spp., (Nematoda: Rhabditidae), a potential entomopathogenic nematode from vermicompost. Nematology. 12:121–135. Zeinat KM, El-Sayed SA, Radwan TEE, El-Wahab, GSA. 2009. Potency evaluation of Serratia marcescens and Pseudomonas fluorescens as biocontrol agents for root-knot nematodes in Egypt. J Appl Sci Res. 4:93–102.
Natural Product Research: Formerly Natural Product Letters Publication details, including instructions for authors and subscription information: http://www.tandfonline.com/loi/gnpl20
Nematicidal activity of microbial pigment from Serratia marcescens a
a
a
Suryawanshi Rahul , Patil Chandrashekhar , Borase Hemant , b
a
ac
Narkhede Chandrakant , Shinde Laxmikant & Patil Satish a
School of Life Sciences, North Maharashtra University, Jalgaon, Maharashtra 425001, India b
Applied Parasitology Research Lab, Department of Zoology, J. E. S. College Jalna, Aurangabad, Maharashtra 431203, India c
North Maharashtra Microbial Culture Collection Centre, North Maharashtra University, Maharashtra, India Published online: 04 Apr 2014.
To cite this article: Suryawanshi Rahul, Patil Chandrashekhar, Borase Hemant, Narkhede Chandrakant, Shinde Laxmikant & Patil Satish (2014) Nematicidal activity of microbial pigment from Serratia marcescens, Natural Product Research: Formerly Natural Product Letters, 28:17, 1399-1404, DOI: 10.1080/14786419.2014.904310 To link to this article: http://dx.doi.org/10.1080/14786419.2014.904310
PLEASE SCROLL DOWN FOR ARTICLE Taylor & Francis makes every effort to ensure the accuracy of all the information (the “Content”) contained in the publications on our platform. However, Taylor & Francis, our agents, and our licensors make no representations or warranties whatsoever as to the accuracy, completeness, or suitability for any purpose of the Content. Any opinions and views expressed in this publication are the opinions and views of the authors, and are not the views of or endorsed by Taylor & Francis. The accuracy of the Content should not be relied upon and should be independently verified with primary sources of information. Taylor and Francis shall not be liable for any losses, actions, claims, proceedings, demands, costs, expenses, damages, and other liabilities whatsoever or howsoever caused arising directly or indirectly in connection with, in relation to or arising out of the use of the Content. This article may be used for research, teaching, and private study purposes. Any substantial or systematic reproduction, redistribution, reselling, loan, sub-licensing, systematic supply, or distribution in any form to anyone is expressly forbidden. Terms &
Downloaded by [University of Newcastle (Australia)] at 10:11 03 September 2014
Conditions of access and use can be found at http://www.tandfonline.com/page/termsand-conditions
Natural Product Research, 2014 Vol. 28, No. 17, 1399–1404, http://dx.doi.org/10.1080/14786419.2014.904310
SHORT COMMUNICATION Nematicidal activity of microbial pigment from Serratia marcescens Suryawanshi Rahula, Patil Chandrashekhara, Borase Hemanta, Narkhede Chandrakanta, Shinde Laxmikantb and Patil Satishac*
Downloaded by [University of Newcastle (Australia)] at 10:11 03 September 2014
a
School of Life Sciences, North Maharashtra University, Jalgaon, Maharashtra 425001, India; Applied Parasitology Research Lab, Department of Zoology, J. E. S. College Jalna, Aurangabad, Maharashtra 431203, India; cNorth Maharashtra Microbial Culture Collection Centre, North Maharashtra University, Maharashtra, India b
(Received 6 January 2014; final version received 11 March 2014) Ineffectiveness of available nematicides and the high damage caused by plant-parasitic nematodes result in the urgent need to find some natural remedy for their control. Bioactivity of the pigment extracted from Serratia marcescens was screened for controlling nematodes at their juvenile stage. Test pigment was found effective against juvenile stages of Radopholus similis and Meloidogyne javanica at low concentrations (LC50 values, 83 and 79 mg/mL, respectively) as compared with positive control of copper sulphate (LC50 values, 380 and 280 mg/mL, respectively). The pigment also exhibited inhibition on nematode egg-hatching ability. Characterisation of extracted pigment with TLC, FTIR, HPLC, HPTLC and spectroscopic analysis confirmed the presence of prodigiosin as a bioactive metabolite. Considering the sensory mechanism of pathogen recognition by nematodes, the use of microbial secondary metabolites can be effective for nematode control rather than using whole organism. Keywords: pigment; nematode; Radopholus similis; Meloidogyne javanica; Serratia
1. Introduction Plant-parasitic nematodes are worm-shaped microscopic animals. Nematodes may cause negligible injury or even total destruction of plant material. Mode of damage to plants by nematodes can be different, it may be reducing or modifying root mass, may act as predisposing agents; for example spread of bacterial canker, they make plants more susceptible to certain fungal pathogens such as Verticillium wilt (Frank 1992) and can cause hormonal imbalance in the root. In addition, nematodes act as vectors for viral infections in plants (Brown et al. 1995), for example both tomato ringspot virus and tobacco ringspot virus are transmitted from infectious plant to normal plant by a common species of dagger nematodes (Xiphinema spp.). Chemical nematicides such as 1,2-dichloropropene, 1,2-dichloropropane, ethylene dibromide, methyl bromide, chloropicrin, metam sodium, dazomet, methyl isothiocyanate, carbamates and organophosphates have been proved to be ecologically unfavourable due to various reasons (Schneider et al. 2003). Environmental problems associated with the use of chemical nematicides have led to a sense of urgency to investigate alternative methods for nematode management (Kerry 2000). Micro-organisms can inhibit plant-parasitic nematodes by use of secondary metabolites, enzymes and toxins. Their manifestation may include suppression of nematode reproduction, egg hatching and juvenile survival, or direct killing of nematodes (Siddiqui & Mahmood 1999).
*Corresponding author. Email: [email protected] q 2014 Taylor & Francis
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Bacteria exhibiting nematicidal effects include members of genera Rhizobium (Neipp & Becker 1993), Enterobacter (Duponnois et al. 1999), Actinomycetes (Yavuzaslanoglu et al. 2011), Burkholderia (Meyer et al. 2001), Serratia (El-Sherif et al. 1999), Chromobacterium (Don et al. 1997), Azotobacter, Bacillus, Clostridium, Corynebacterium and Methylobacterium (Prabhu et al. 2009). Rhizobacteria are the only group of micro-organisms in which biological nematicides have been reported, besides the nematophagous fungi and Actinomycetes (Baoyu et al. 2007). Most thoroughly studied organism for nematicidal activity is Bacillus subtilis (Siddiqui & Shaukat 2002). Bio Yield (Gustafson LLC, Plano, TX, USA) and BioNem are few commercially available products based on Bacillus nematicidal activity (Giannakou & Prophetou 2004). Few Cry proteins from Bacillus thuringiensis are also known for nematicidal potential. Serratia marcescens, a Gram-negative bacterium, has been reported to produce cellassociated red-coloured pigment called prodigiosin (Carbonell et al. 2000). This deeply studied pigment exhibits inhibitory activity against Gram-positive micro-organisms, antifungal (Chandni et al. 2012), antiprotozoal (Genes et al. 2011), antimalarial, immunosuppressive (Tsuji et al. 1990), anticancer (Diaz et al. 2001) and insecticidal (Chandrashekhar et al. 2011) activities. These properties make prodigiosin a powerful research tool for screening against a range of plant and animal pathogens. S. marcescens as a whole organism has also been reported as a pathogenic strain for plant-parasitic nematodes. Considering the antibiotic potential of prodigiosin, it was screened against the juveniles of plant-parasitic nematodes associated with banana and brinjal. Crop losses caused by nematodes to these plants are very high. Worldwide, an average annual yield loss was estimated to be about 20% (Sesser & Freakman 1987). Toxicity studies of prodigiosin on agriculturally important micro-organisms (AIMs) have also been conducted to ensure eco safety aspects of pigment in actual field conditions. The pigment was characterised using different techniques. 2. Results and discussion After incubation of Serratia culture, the pigment was extracted from the biomass with acidified methanol. The crude extract was evaporated till complete drying. Furthermore, the dried pigment was purified by TLC and then quantified on dry weight basis. A total of 50 mg pigment was produced. In TLC analysis, the extract displayed an Rf value (0.87) similar to that of standard prodigiosin (Sigma-Aldrich, St. Louis, MO, USA) with butanol:hexane (2:1) as a solvent system (Figure S1). Spectroscopic studies indicated maximum absorbance at 535 nm (Figure S2), which is in accordance with results for prodigiosin from S. marcescens. The HPLC and HPTLC analyses revealed single peak at 536 and 535 nm, respectively, with a retention time of 13.4 min in HPLC (Figure S3). In FTIR analysis (Figure S4), red pigment from S. marcescens revealed strong absorption at 3516 per cm (NZH) and 2997 per cm (aromatic CZH), 1668 per cm (aromatic CvC) which suggests the presence of respective bands; prodigiosin exhibits similar absorptions in CHCl3 at 1630, 1602 per cm. Fingerprint region of the same displayed distinctive intensity band at 1668 per cm (aromatic Cv0), 1039, 1018 (CZO, CZN) carboxylic groups, 953, 899, 703 per cm (CZH) alkanes, 1313 (nitro compounds), 1434, 1410 (CZH) alkenes. Transmission pattern of the pigment was similar for the functional groups present in prodigiosin. Characterisation studies such as TLC, FTIR, HPLC, HPTLC and spectroscopic analysis confirmed the presence of prodigiosin in the sample. These results are in accordance with Song et al. (2006). Purified pigment was evaluated for nematicidal activity against plant-parasitic nematodes Radopholus similis and Meloidogyne javanica. In larvicidal bioassay, the pigment exhibited highly toxic action against juveniles of R. similis and M. javanica with LC50 values of 83, 79 mg/ mL and LC90 values of 272, 182 mg/mL, respectively. Copper sulphate was used as a positive
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control; it displayed LC50 values of 380, 280 mg/mL and LC90 values of 1810, 1040 mg/mL against R. similis and M. javanica, respectively (Figure 1 and Table 1). Same concentrations of pigment when tested for nematode egg hatching exhibited complete inhibition of the hatching ability of nematode eggs, and no juveniles were observed to be coming out of the eggs till four weeks for all tested concentrations (data not shown). Larvicidal assay demonstrated that the LC50 values presented by the pigment are significantly low when compared not only against positive control (copper sulphate) (Table 1) but also against synthetic pesticides such as chlorpyrifos, carbosulfan and deltamethrin, which presents LC50 values of 19.4, 25.3, and . 40 mg/mL, respectively, against Meloidogyne spp. (Wiratno et al. 2009). The concentration- and time-dependent study of prodigiosin and copper sulphate revealed that the prodigiosin possessed high nematicidal potential than copper sulphate at very low concentration and in a very short time (Figure 1). Our observation supports the pigment of S. marcescens as a stronger nematicidal candidature compared with copper sulphate and synthetic pesticides. Toxicity studies of prodigiosin against AIMs such as Bradyrhizobium japonicum, Azotobacter vinelandii and Trichoderma viride indicated normal growth of these AIMs in the presence of high prodigiosin concentration (500 mg/mL). Our observations suggest that the use of prodigiosin even at high concentrations is safe for agriculturally important flora of the soil. Insecticidal properties of S. marcescens are well known including the genera Anomala, Costelytra and Phyllophaga (Nunez et al. 2008); prodigiosin has been demonstrated to decrease egg-hatching ability in budworm Lepidoptera. It has also been reported to inhibit different stages of mosquito larvae (Aedes aegypti and Anopheles stephensi) (Chandrashekhar et al. 2011). Serratia can be used to control the diamondback moth, Plutella xylostella (Jeong et al. 2010). In another study, S. marcescens was found to reduce the development of root-knot nematode Meloidogyne incognita by high percentage reduction in the number of the juveniles in soil ranging from 77.2% to 84.4% in situ (Zeinat et al. 2009). S. marcescens was also used in
Figure 1. [A] Percent survival of juveniles (a) R. similis and (b) M. javanica against pigment, from Serratia with respect to time of incubation. [B] Percent survival of juveniles (c) R. similis and (d) M. javanica against positive control, from Serratia with respect to time of incubation.
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Table 1. Nematicidal activity of pigment prodigiosin from S. marcescens. Nematode
Test product
LC50 ^ SD (mg/mL)
LC90 ^ SD (mg/mL)
R. similis
Prodigiosin Positive control Prodigiosin Positive control
83 (^0.6) 380 (^8) 79 (^0.5) 280 (^4)
272 (^2.5) 1810 (^15) 182 (^7) 1040 (^10)
M. javanica
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Notes: Positive control, copper sulphate; SD, standard deviation; LC50, lethal concentration that kills 50% of juveniles; LC90, lethal concentration that kills 90% of juveniles. Level of significance P , 0.05.
agricultural field for the suppression of M. incognita to increase the yield of sunflower (Ali 1996). An enzyme, chitinase, from Serratia spp. is also reported to cause premature egg hatching of Meloidogyne helpa (Mercer et al. 1992). The effect of Serratia on nematodes appears to be strain specific as some researchers have reported symbiotic association of Serratia nematodiphila with nematodes such as Heterorhabditidoides chongmingensis. Several entomopathogenic nematodes such as Oscheius chongmingensis, Oscheius carolinensis and Caenorhabditis briggsae employ insect pathogenic bacteria such as Serratia to kill insects (Abebe et al. 2010; Ye et al. 2012; Torres et al. 2011). Though reports are available for the direct use of Serratia spp., as a biocontrol agent, its use may be limited since this approach could be pathogenic to animals as well (Jeong et al. 2010). Sensory mechanism for pathogen recognition has been reported in nematodes such as Caenorhabditis elegans. They are able to detect the pathogenic bacterium such as S. marcescens and can avoid the organism from entering inside the body, based on the production ability of the cyclic lipodepsipentapeptide serrawettin W2 (Elizabeth et al. 2007). Concerning this mechanism, the use of secondary metabolites rather than bacterial cells could be an effective way to control the growth of plant-parasitic nematodes. Prodigiosin from Serratia spp. possesses proton-sequestering ability (Roser et al. 2007), which affects intracellular pH gradient. Prodigiosin also affects the mitochondria by exerting an uncoupling effect on the electron chain transport of protons to mitochondrial ATP synthase; it causes reduction in ATP production without decreasing the oxygen consumption. Few other studies have also reported prodigiosin as a proton-pump inhibitor (Sato et al. 1998; Matsuya et al. 2000). Action of prodigiosin on the parasite Trypanosoma cruzi is found to be exerted by structural and functional disruptions of mitochondria, which might escort the parasites to an apoptotic-like cell death process (Genes et al. 2011). 3. Conclusion The use of secondary metabolites can be an effective way for nematode inhibition rather than the use of whole organism. Low concentrations of tripyrrol pigment from S. marcescens have been found to inhibit the juvenile stage of nematodes (R. similis and M. javanica); the LC50 value of the pigment is 5- to 10-fold less than other pesticides. The non-toxicity of prodigiosin against agriculturally important flora suggests the eco safety of the pigment. In order to explore the nematicidal potential of prodigiosin, field studies are required to confirm the infield activity. Specific reason for nematicidal action of prodigiosin merits further research. Supplementary material Experimental details relating to this article are available online, alongside Figures S1– S5.
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Acknowledgements We are thankful to Rajiv Gandhi Science and Technology Commission, Mumbai, Government of Maharashtra, India for financial assistance. We are also thankful to North Maharashtra Microbial Culture Collection Centre for providing microbial cultures.
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