Studies on the occurrence of endophytic bacteria in Angelonia salicariaefolia

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1 Malaya Journal of Biosciences 2015, 2(1):36-41 ISSN print / online Malaya Journal of Biosciences RESEARCH ARTICLE Open Access Full Text Article Studies on the occurrence of endophytic bacteria in Angelonia salicariaefolia M. Duraisankar 1 and S. Balakrishnan 2 1 Department of Botany and Microbiology, AV.V.M. Sri Pushpam College, Poondi, Thanjavur 2 Professor and Head, Department of Botany and Microbiology, AV.V.M. Sri Pushpam College, Poondi, Thanjavur. * For correspondence dsankar2012@gmail.com. Article Info: Received 14 Jan 2015; Revised: 22 May 2015; Accepted 27 May 2015 ABSTRACT Endophytic bacteria seems to have a ubiquitous existence in most, if not all, higher plant species without causing disease symptoms. Therefore, they seems to be ideal to complement certain metabolic properties of their host plant, such as the fixation of nitrogen or the detoxification of contaminants. The endophytes are sometimes neutral in function with plant in the initial stages and later become active during plant development. In most cases the endophytes are isolated from root free soil as they are ecologically dependent on the host plant. The present study has been undertaken, to isolate endophytic bacteria from Angelonia salicaraefolia, to identify the isolated bacteria using biochemical tests, to study the effect of plant extracts on other harmful pathogens. Keywords: Endophytic bacteria, Isolate and identification, Anti-bacterial activity 1. INTRODUCTION Endophytic are organisms that live inside plants. They complete their life cycle or part of their life cycle within the plant. Microbes such as bacteria, fungi, cyanobacteria are the most common organisms which lead an endophytic mode of life. The endophytic occurrence of these organisms has been reported from various parts of the plant such as root, stem, leaves etc. The endophytes confer benefits to the plant and vice versa. There are different views about the endophytic association of bacteria within living plants. The endophytic association of bacteria expresses a natural benefit thereby sharing and getting nutrients and residence from plants which intern provides substances to improve plant growth [1]. From the evolutionary point of view it is considered that the endophytic bacteria are originally derived from plant pathogens and they would have - lost their virulence due to extended growth period within the plant. As a result, the endophytic bacteria are unable to express genes to diseases [2]. However the plant pathologist feels that through the endophytic bacteria influence the host plant towards the direct antagonism or niche, exclusion of the pathogen by induction of systemic resistance and increasing plant tolerance towards biotic stresses. The endophytes are sometimes neutral in function with plant in the initial stages and later become active during plant development. In most cases the endophytes are isolated from root free soil as they are ecologically dependent on the host plant. They colonize mostly in root cortex and to a lesser extent in the vessels. In the initial stages, it is believed that the endophytic bacteria are supporting their host plant Studies on the occurrence of endophytic bacteria in Angelonia salicariaefolia Copyright 2015 MJB 36

2 by means of supplying nitrogen. It is proved now adays that they can support the host plant by providing other essential substances and nutrients also and further product the hosts from pest attack [3]. Further it is well established that the endophytic association in many cases are responsible for the presence of novel metabolites in the host tissues. Therefore the study of endophytes is in the development of tolerant species, nitrogen fixing efficiency of plants, bio-herbicides and pharmacological agents [4]. The scientists have paid considerable attention on the endophytic microbes, but their studies are mainly focused on economically important crop plants. 2. MATERIALS AND METHODS 2.1. Sample collection In the present study an attempt has been made to find out the occurrence of endophytic bacteria inside the stem of Angelonia salicariaefolia. The plants were collected from Arul Nursery, Thanjavur, during January The plants raised in pots were transported to laboratory and hand sections of the stem were taken with the help of a sharp blade. The sections were observed under microscope and photomicrographs were taken Systematic position of Angelonia salicaraefolia (plate 1) (Bentham &hooker, ) Class Subclass Order Family Genus Species : Dicotyledones : Gamopetalae : Personales : Scrophulariaceae : Angelonia : Salicariaefolia 2.3. Microbial isolation and identification To analyze endophytic microflora of plant, Angelonia salicaraefolia the stem were hand sections taken with the help of a sharp blade Stem samples were used for isolation of endophytic bacteria the stem were washed and shaking for one hour with 100 ml of sterile distilled water. An aliquot of 1 ml from stem wash were plated separately LB medium- Peptone 5 g/l, Beef Extract 3 g/l, Agar 15 g/l for bacterial isolation. For endophytic microbial isolations, host stem were submerged in 70% ethanol (v/v) for 1 min to wet the surface, followed by surface disinfection for 3 min in 2% NaClO2. Afterward sample materials were rinsed 2-3 times with sterile distilled water, transferred to sterile filter paper and dried in laminar flow. About 2 g of sterilized sample (stem) were ground into paste with 20 ml of sterilized water. For 1 ml of paste solution was added on the LB medium and incubated at 37 C. Bacterial strains were identified including pigment, colony form, elevation, margin, texture and opacity [5]. In addition, bacterial strains were tested with respect to Gram reaction and biochemical characteristics [6] Biochemical test Gram staining The bacterial culture growing on Nutrient agar was smeared on a glass slide. The smear was treated with crystal violet for 30 seconds, and washed with distilled water. The smear was maintained in iodine solution for 30 seconds and washed with distilled water. Finally the slide was kept in safranine for 30 seconds, and again washed with distilled water and examined under the microscope. It was observed as single celled, Gram negative, rod shaped bacteria Carbohydrate fermentation test A loopful culture was inoculated in the 3 tubes containing fermentation broth: trypticase-10g, Carbohydrate-5.0g, Sodium chloride 15.0g, Phenol red g, Distilled water-100ml using lactose, glucose sucrose separately and incubated at 35 0 c for 24 hours. A control was also maintained without inoculating the bacteria Citrate utilization test MR-VP broth was prepared and filled in test tubes, Simmons Citrate Agar: Ammonium dihydrogen phosphate - 1.0g, Sodium chloride - 5.0g, Magnesium sulphate - 2.0g, Bromothymol blue - 0.8g, Agar- 15.0g, and Distilled water-1000ml. Loopful culture was inoculated in the test tubes and incubated at 35 0 c for 48 hours. To this, 5 drips of methyl red indicator were added. The colour change was observed from red to rose and this was considered as a positive result. A control was also maintained Growth and maintenance of test microorganism for Antimicrobial studies Bacteria cultures of Escherichia coli, Staphylococcus aureus were obtained from the culture collection center, Department of Microbiology, A.V.V.M Sri Pushpam collage Thanjavur, India, were used for antimicrobial test organisms. The bacteria were maintained on nutrient broth (NB) at 37 º c. 37

3 2.5. Preparation of inoculums The gram positive (Staphylococcus aureus) and gram negative bacteria (Escherichia coli) were precultured in nutrient broth overnight in a rotary shaker at 37 0 c centrifuged at 10,000 rpm for 5 min pellet was suspended in double distilled water and the density was standardized spectophotometrically at 610 nm Antibacterial activity Angelonia salicaraefolia the stem was taken and the pith region was removed. The remaining portion was ground separately in a mortar and pestle to get aqueous extracts were tested by the disc diffusion method. The extract was mixing with Dimethyl sulphite different concentration 10 µg, 100 µg, 200 µg, 400 µg, 800 µg, 1000 µg The test microorganisms were seed into respective medium by spread plate 10µl(106 cells/ml) with the 24 h cultures of bacteria growth in nutrient broth. After solidification, the filter paper discs (5mm in diameter) in extract were placed on the on test organisms seeded plates Escherichia coli (Table-1), Staphylococcus aureus (Table-2), and Streptomycin sulphate (10µl/ ml) used as positive control and methanol solvent (100µl/ml) used as negative control the antibacterial assay plates were incubated at 37 0 c for 24hrs the diameters of in the inhibition zone were measured in mm. bacterial cells in large numbers in paraenchymatous pith cells. Thus the endophytic occurrence of bacteria was observed as encapsulated one with many bacterial cells in the vesicle. A vesicle was seen around the bacterial cells, which leaves a space from the cell wall. The vesicles are visible only in the thick hand sectioning (Figure -3). The bacteria was isolated and grown in nutrient agar medium (Figure-4). Examination of morphological characteristics showed that single and rod shaped cells (Figure-5). The cells are motile. Biochemical tests showed sucrose, glucose and lactose as positive and in MR test positive result obtained for citrate utilization and gram staining result negative was observed. Figure 1. Angelonia salicaraefolia grown in pot. 3. RESULTS Angelonia salicariafolia is grown in pots (Figure.1) as an ornamental plant in gardens and houses. It is a tropical shrub that preferably grown in a green houses in the temperature range of 60 to 65 0 c. Obtaining seeds of the plant is rare and therefore the plant is multiplied by vegetative propagation method. It requires moist soil for its growth. This plant produces beautiful variegated flowers with full bloom in late winter or early spring season. Stem is herbaceous, quadrangular in outline. Flowers are bisexual, irregular, and strongly zygomorphic with blipped corolla. The corollary tube is perigynous, alternate with the lobes. The gynoecium consists of a single compound pistil of two carpels, a single style and a superior, asymmetric ovary with axile ovules. The fruit is usually a capsule. The fine cross section of the stem revealed the anatomy with three distinguished parts viz., epidermis, cortex and stele. Cortex consists of parenchymatous cells. Stele consists of vascular tissues and pith (Figure.2). Microscopic examination of the section further revealed the occurrence of There are characteristic features of the genes Bacillus, and therefore the bacterial isolate is categorized as Bacillus group. The colonies were white in colour and grow in temperature of 28 0 c with a moderate growth in nutrient agar media with undulate margin. In broth they formed pellicles with cloudy mass. As the Angelonia plant has the endophytic bacteria it was evaluated for antimicrobial property against (Figure-7) Eschericha coli, Staphylococcus aureus. 38

4 The stem extract, obtained after removing pith region, inhibited the growth of However, when endophytic isolate was placed in a nutrient agar plate containing antibacterial activity inhibition zone was not formed. This shows that the plant extracts are responsible for arresting the growth of Staphylococcus aureus and E.coli (Table-1 & 2) but not the endophytic bacteria. The endophytic occurrence of bacteria was reported for the first time in Angelonia. However it requires a detailed study on the reasons for the occurrence, the nature of the association etc. Figure 2. Photomicrograph showing the occurrence of bacteria in pith cells of stem. exudates from the stem inhibit the growth of Eschericha coli, Staphylococcus aureus, but not the endophytic isolate. This further indicates the close relationship between the Angelonia salicariaefolia and the Bacillus sp will be beneficial to both the plants. However, further investigation is necessary to reveal the exact relationship between these two organisms. The presence of vesicles inside the cells may be for giving an O 2 free anaerobic atmosphere to the bacterial system to do the function of N 2 fixation. The potentiability of Angelonia salicariaefolia plant to horbour the endophytic bacteria in the stem tissues is very interesting and it has to be studied in various angles to get answer for the questions with reference to their occurrence, function, mode of entry multiplication host-endophytic interactions etc. [10,11]. 4. DISCUSSION Endophytic occurrence of bacteria is not a common feature expect the occurrence of Rhizobium in stem and root nodules of leguminous plants. In some cases the bacteria enter into the plant parts especially leaf and cause disease to the plants. The present study, revealed the occurrence of endophytic bacteria similar to that of the genus Bacillus in the pith regions of the stem, Angelonia salicariaefolia. There are two system of bacterial association explained, with the plants. In the first group, the symbionts rhizobia are capable of forming effective nodules both on the roots and stems of the host plant and create ideal nitrogen fixing system in the host plant. Both the host plant and the Rhizobium lead a symbiotic life [7-9]. In the second group, the pathogens, the disease causing bacteria enter into the plant tissue and cause damage to the plant tissues thereby causing decrease in productivity and ultimately the host plant is suffering for its existence. But in the present study the occurrence of Bacillus like bacteria in the stem tissues appears to help the plant for its better growth of the host Angelonia salicariaefolia as it grew better with the endophytic association of these bacteria. Further, being the soft tissue, the pith would have allowed the entry of the bacteria and facilitated its survival, as the plants are multiplied through vegetative means. It is also interesting to note that the Figure 3. Photomicrograph showing the presence of vesicle inside the pith cells Figure 4. Isolation of endophytic bacteria from the stem section using nutrient medium and pure culture obtained. 39

5 Table.1 Anti-bacterial activity assay with Staphylococcus aurous Concentration Zone of Percentage of inhibition inhibition 10 µg/ml 4mm µg/ml 8mm µg/ml 9mm µg/ml 12mm µg/ml 16mm µg/ml 22mm µg/ml 28mm 100 Figure 7. Formation of inhibition zone by plant extract against staphylococcus aureus and Gram staining of Staphylococcus Gram negative coccus. Table.2. Anti-bacterial activity assay with Escherichia coli Concentration Zone of inhibition Percentage of inhibition 10 µg/ml 4mm µg/ml 5mm µg/ml 8mm µg/ml 12mm µg/ml 16mm µg/ml 20mm µg/ml 26mm 100 Figure 5. Pure culture of endophytic bacteria (A) ; (B) Gram staining in endophytic bacteria 5. CONCLUSION In the present study an attempt has been made to find out the occurrence of endophytic bacteria inside the stem of Angelonia salicariaefolia. Abundant of bacterial cells were observed inside the parenchyma cells especially in the pith regions. A vesicle is formed inside the pith cells in which the bacteria are living. The bacterium was isolated by using Nutrient Agar medium. They grew moderately in the medium. Biochemical tests and morphological examination resulted Bacillus like characteristics, of the bacteria. The occurrence of endophytic Bacillus like bacteria is reported for the first time in Angelonia salicariaefolia stem. The stem also showed inhibitory effects on the growth of Escherichia coli, Staphylococcus aureus. Further studies are required to review the nature of endophytic association of bacteria with host plant and nature of substances produced from the stem which inhibited the growth of Staphylococcus aureus. Acknowledgement Figure 6. A Lactose-postive; B: Glucose-positive; C: Citrate utilization positive and D: MR-VP test. The authors are grateful to the Department of Botany and Microbiology, AV.V.M. Sri Pushpam College, Poondi, Thanjavur for supporting this research work. Conflict of Interest The authors declare that they have no conflicts of interest. References 1. Johnson, H.W., Response to nodules bearing living in endophytic association. Nature, 183: Boyer, H. W., and Roulland-Dussoix, D A complementation analysis of the restriction and modification of DNA in Escherichia coli. Journal of Molecular Biology. 41:

6 3. Anderson, J.M., The review of the genes involved in nitrogen fixation. Symbiosis in Science, 260: Bacon, B.L.and Charles Ulenn, Isolation in plant to detection, and culture endophytic bacteria and defoned are those organisms. Environmental Microbiology. 165: Smibert, R.M. and Krieg, N.R. (1981) General Characterization. In: Gerdhardt, P., Murray, R.G.E., Costilow, R.N., Nester, E.W., Wood, W.A., Krieg, N.R. and Phillips, G.B., Eds., Manual of Methods for General Bacteriology, American Society for Microbiology, Washington, DC, Holt, J., N. Krieg, P. Sneath, J. Staley and S. Williams Bergey's Manual of Determinative Bacteriology (9 ed.), Williams and Wilkins, Baltimore, USA. 7. Elberltagy, A., Endophytic colonization and in plants nitrogen fiation in wild rice spricies. Applied Environmental Microbiology, 67: Monigue, A Endophytic plant growth promoting PGR bacteria in carrots, Canadian society for Horticultural Science, Morisahi, H., Isolation and characterization of endophytic bacteria from wild and traditionally cultivated rice varieties. Soil science Plant Nutrients, 46(4): Timothys Schubert, K., Serratia marcescens endophytic in plants, used as a plant disease bio- control agent. Phytopathology, 9: Yuhashi, G.k., Rhizobitoxine production by Bryadyrhizobium elkanii enhances nodulation and competitiveness on macroptitium. Applied Environmental Microbiology, 66: