A study on the potential of endophytic bacteria as biocontrol agent

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1 SIRJ-BES Volume 1 Issue 10 (2014) ISSN Scrutiny International Research Journal of Biological and Environmental Science (SIRJ-BES) A study on the potential of endophytic bacteria as biocontrol agent Priscilla Helen Christy S 1 and Sudha S.S 2 1 School of Biological Sciences, C.M.S College of Science and Commerce, Coimbatore. 2 Department of PG & Research in Microbiology, Dr. N.G.P College of arts and Science, Coimbatore. Article history: Submitted 2 October 2014; Accepted 28 October 2014; Available online 15 December 2014 Abstract Pathogenic microorganisms affecting plant health are a major and chronic threat to food production and ecosystem worldwide. As agricultural production intensified over the past few decades, producers became more and more dependent on agrochemicals as relatively reliable method of crop protection helping with economic stability of their operations. Plant pathogenic fungi are of major concern problem in agriculture. Biocontrol methods are safe, cost effective and eco-friendly. In present study, HCN and siderophore producing Pseudomonas aeruginosa was selected. Sclerotina sclerotiorum and Fusarium oxysporum were isolated from infected tomato plant and grown on PDA MEDIUM and characterized. Antagonistic activity of Pseudomonas against Sclerotina sclerotiorum was evaluated. All Pseudomonas aeruginosa strain inhibited the growth of Sclerotina sclerotiorum by 3mm inhibition zone as compared to control. Pseudomonas strain showed maximum inhibition. In vitro study revealed that Pseudomonas strain effectively reduced the growth of Sclerotina sclerotioum. Key words: Antagonistic, Biocontrol, PDA, Pseudomonas aeruginosa, Sclerotina sclerotiorum Corresponding author Priscilla Helen Christy. S. Asst professor, School of Biological Sciences C.M.S College of Science and Commerce, Coimbatore Introduction Soil borne plant pathogens can significantly reduce yield and quality in vegetable crops. These pathogens are particularly challenging because they often survive in soil for many years and each vegetable crop may be susceptible to several species. However, increasing use of chemical inputs causes several negative effects, i.e., development of pathogen resistance to the applied agents and their non target environmental impacts. Furthermore, the growing cost of pesticides, particularly in less-affluent regions of the world, and consumer demand for pesticide-free food has led to a search for substitutes for

2 Prisilla & Sudha / SIRJ-BES 1:10 (2014) these products. Biological control is thus being considered as an alternative or a supplemental way of reducing the use of chemicals in agriculture. Mangroves are highly productive ecosystems which cover roughly 60-75% of the world s tropical coastlines. Distributed over more than 112 countries with a total area near 181,000km 2 productivity (Shakilabanu et al., 2012); Endophytic bacteria have been found in virtually every plant studies, where they colonize the internal tissues of their host plant and can form a range of different relationships including symbiotic, mutualistic and commensalistic and trophobiotic. Endophytes can also be beneficial to their host by producing a range of natural products that could be harnessed for potential use in medicine, agriculture or industry. White mold affects a wide range of hosts. It is known to infect 408 plant species. Its diverse host range and ability to infect plants at any stage of growth makes white mold a very serious disease. The fungus can survive on infected tissues, in the soil, and on living plants. It affects young seedlings, mature plants, and fruit in the field or in storage (Bardin and Huang, 2001) Biocontrol of plant disease involves the use of an organism or organisms to reduce disease. (Glick et al., 1995); Reports suggest that Fluorescent Pseudomonas inhibited the growth of pathogenic fungi such as Fusarium solani, Pythium ultimum and Rhizoctonia solani. Recently, Pseudomonas fluorescens was used as bio-control agent for controlling broccoli root rot disease caused by Pythium ultimum and Rhizoctonia solani pathogens. (Adams and Ayers, 1979); Biological control of plant pathogens by antagonistic microorganisms is a potential non-chemical means and is known to be a cheap and effective eco-friendly method for the management of crop diseases (Pal and gardner, 2006). Materials and Methods Sample collection and Location Samples were collected from mangrove forest situated in chettuva backwaters, Trissur, Kerala. Healthy leaf, stem and root samples of mangrove species Rhizophora mucronata were collected in sterile bags and transported to the laboratory aseptically. Isolation and characterizing of endophytic bacteria Collected samples were washed in running tap water followed by 70 % ethanol, 2 % Sodium hypochlorite and distilled water respectively and air dried under a laminar flow hood for surface sterilization. The outer tissues were removed and the inner tissues were macerated with distilled water using mortar and pestle. 1 ml of macerated sample was serially diluted and 100µl of each dilution was inoculated on nutrient agar medium by spread plate method. Plating was done in triplicates and incubated at 37º C for 48 hours. Identification of endophytic bacteria was carried out by the routine tests like Gram s staining, Spore staining, Biochemical characterization which includes Indole test, Methyl red test, Voges- proskauer Test, Citrate Utilization Test, Oxidase test, Catalase test, Triple sugar iron test Motility Test etc. Culture Method The selective medium for Pseudomonas is Cetrimide agar medium. The bacterial isolate was streaked (Quadrant streak) on to sterilized Cetrimide agar plate and are incubated at 37 C for 24 hours. 8

3 Prisilla & Sudha / SIRJ-BES 1:10 (2014) Screening and identification of pathogenic fungi The sample was collected from Tamil Nadu Agriculture University (TNAU).The organism which was isolated from tomato plant cause white mold disease called as Sclerotinia sclerotiorum, which shows white color and Fusarium which cause wilt disease. Identification of the fungi was carried out using Lactophenol cotton blue staining technique. The cell morphology was recorded with respect to spore chain morphology, hyphae and mycelium structure. Sub culturing of pathogen in medium Sub culturing of fungi were done by slide culture technique using sterile forceps the fungi was cut along with agar piece then transferred to sterile PDA plates and incubated for 3 to 5 days at room temperature. Antagonistic activity of Pseudomonas against Sclerotinia sclerotiorum Antagonistic activity of Pseudomonas strain was tested against Sclerotinia sclerotiorum by using dual culture technique. It can be done in 2 ways: a) Method I: Five-day-old mycelial discs (5 mm diameter) of Sclerotinia sclerotiorum were placed at four corners on the modified King s B medium by including 2% sucrose. Exponentially grown Pseudomonas strain was spotted in the centre of agar plates and incubated at 28 ± 1 C for five days (king s et al-1954). Inhibition in radial growth of test fungus was measured. One fungal disc was transferred at centre of control plates and radial growth was measured. b) Method II: Pseudomonas strain was swabbed on to a King s B agar plate using a sterile swab. The fungal culture was inoculated on the centre of the plate and was incubated at 28 ± 1 0 C for 5 days. Results Collection of sample The healthy plant samples of mangroves were collected in a clean leak proof polythene bag. It was then transferred to biotechnology lab, CMS College of Science and Commerce, Coimbatore and maintained in a laboratory tray containing water. Isolation & Identification of endophytic bacteria from the healthy plant The incubated NA plates showed mixed colonies of bacteria after 1 day of incubation. A particular green coloured colony was observed and this was chosen for further studies (Fig 1; Fig 2-3). The isolated bacterial cultural were identified as Pseudomonas sp by the microscopic examination, biochemical characteristics and cultural characteristic observations. Microscopic Observations showed Gram negative (Fig 4), Non spore forming, motile cells. The biochemical test of the bacterial isolate was tabulated in (Tab1; Fig 6-12). 9

4 Prisilla & Sudha / SIRJ-BES 1:10 (2014) Table No. 1: Biochemical Test of Bacterial isolate Culture Method Sl. No Test Results 1. Indole _ 2. Methyl red _ 3. Voges-proskauer _ 4. Simmon citrate + 5. Catalase test + 6. Oxidase test + 7. TSI test + Green colour colonies were observed on Cetrimide agar plate along with the swarming motility (Fig 5). Cultivation of pathogen fungi The pathogenic fungi that collected from TNAU were reconfirmed by microscopic and cultural characteristic observation on PDA are listed (Table 2; Fig 13 & 14) Table No. 2: Characteristics of Fungal Pathogens Fungal pathogen Fusarium oxysporum Sclerotinia sclerotiorum Microscopic Observation Conidiogenous cells hyaline, enteroblastic, mono or polyphialidic. Fusarium species produce several types of conidia. Microconidia hyaline, 0-1 or septate, small, macroconidia hyaline, curved, phragmospores, with a foot cell bearing, some kind of heel. Hyaline, septate, branched and multinucleate, and their colors changed from white to dark as melanin accumulates. Hyphae tend to form sclerotia. Asci are cylindrical sac-like zygote cells and are rowed in the hymenial layer. Each ascus contains eight hyaline, ellipsoid binucleate ascospores [(4~6) μm (9~14) μm]. Antagonistic activity Method-1 (zone of inhibition) 3 mm Method -2 (zone of inhibition) 1.5 mm Cultural Characteristics on PDA plate Growth moderate, white, peach, to salmon pink or Violet. At initiation stage, hyphae aggregate to form a white mass, then further aggregate to increase the size of sclerotia; finally, surfaces of sclerotia are delimited, with melanin deposited in peripheral rind cells, and interiors of sclerotia become consolidating. (2~10 mm in diameter). Decolourization of Acid orange 7 by selected bacterial strains. The selected five isolates were checked for their ability to decolourize Acid Orange 7 at an increased dye concentration of 50 mg/l. The decolorization rate was expressed in % degradation and the results are tabulated (Fig 1). Out of the selected isolates, B1 was the best decolourizer with a percentage decolourization of 84.53%, followed by A2, B3, A3 and B2 with an efficiency of 73.31%, 67.26%, 62.32% and 54.65% respectively. 10

5 Figure No. 1: Figure No. 2: Figure No. 3: Figure No. 4: Bacterial Isolate Growth observed Microscopic view Green coloured colony on cetrimide agar media Figure No. 6: Figure No. 7: Figure No. 8: Figure No. 9: Figure No. 10: Indole test Methyl red test Voges proskauer test Citrate test TSI test Figure No. 11: Figure No. 12: Figure No. 13 Catalase test Oxidase test (a) F.oxysporum on PDA plate (b) Microscopic View Figure No. 14: S. sclerotiorum Figure No. 15: S. sclerotiorum (a) PDA plate (b) Microscopic View (a) Control plate (b) Method I (c) Method II

6 Discussion The study was mainly concentrated on Pseudomonas sp and so the green colored colony observed on the nutrient agar plate was chosen for the study which was suspected as Pseudomonas spp. Different tests were performed for the confirmation of Pseudomonas and thus the microscopic observation, culture method and biochemical test confirmed that the bacterial isolate was Pseudomonas spp. Further the species level identification was done by Maldi-tof method. Thus the isolate was found to be Pseudomonas aeuroginosa. The fungal pathogen chosen for the study were Sclerotinia sclerotiorum and Fusarium oxysporum. The microscopic and cultural characteristics were studied for both the pathogens. The antagonistic activity performed by method 1 was found to be more effective when compared to method II. Pseudomonas was found to be a good antagonist against Sclerotinia sclerotiorum when compared to Fuasrium oxysporum. Thus the Pseudomonas aeuroginosa can be used as good biocontrol agent against the white mold in tomato in which the causative agent is Sclerotinia sclerotiorum. Reference Bardin, S. D., Huang, H. C., Research on biology and control of Sclerotinia diseases in Canada. Can. J. Plant Patho., 23: Boland, G. J., Stability analysis for evaluating the influence of environment on chemical and biological control of white mold (Sclerotinia sclerotiorum) of bean. Biol. Control., 9: Deshwal, V. K., Pandey, P., Kang, S. C. and Maheshwari, D. K., Rhizobia as a biological control agent against soil borne plant pathogenic fungi. Ind J Exp Biol, 41: Glick, B. R., Karaturovic, D. M. and Newell, P. C., A novel procedure for rapid isolation of plant growth promoting Pesudomonads. Can J Micobiol, 41: Ligon, J. M., Hill, D. S., Hammer, P. E., Torkewitz, N. R., Hofmann, D., Kempf, H. J., Van Pee, K. H., Natural products with antifungal activity from Pseudomonas biocontrol bacteria. Pest Mag. Sci., 56: Shakilabanu, S., Kanchana, D. and Jayanthi, M., Biodiversity of plant growth promoting Rhizobacteria in mangrove ecosystem International Journal of Pharmaceutical and Biological archives, 3(3):