Rhizobium spp. were isolated from root nodules of 10 cultivated & three wild legume hosts. All the isolated Rhizobia were individually tested for their antibiotic response. Biodiscs of five antibiotics having three different concentrations viz: 0.5%, 1% & 1.5% were tested against the isolated Rhizobia using plate culture method. The affectivity of the antibiotics was revealed in terms of zone formation & was measured in mm

R. Diwan, S. Sharma* and

Journal of Ecobiotechnology

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Journal of Ecobiotechnology 2011, 3(4): 13-15 ISSN: 2077-0464 www.scholarjournals.org www.journal-ecobiotechnology.com 13  Characterisation of Rhizobia on the Basis of Antibiotic Responses  S. Sharma* and  R. Diwan Depatment of Botany, Govt. N.P.G. College of Science, Raipur (C.G.), India Article Info  Abstract Article History  Rhizobium spp. were isolated from root nodules of 10 cultivated & three wild legume hosts. All the isolated Rhizobia were individually tested for their antibiotic response. Biodiscs of five antibiotics having three different concentrations viz: 0.5%, 1% & 1.5% were tested against the isolated Rhizobia using plate culture method. The affectivity of the antibiotics was revealed in terms of zone formation & was measured in mm.  Received : 22-03-2011  Revised : 29-04-2011  Accepted : 30-04-2011  *Corresponding Author  Tel : +91-9977680390  Fax : +91-7714027432   Email:sst2008@gmail.com    ©ScholarJournals, SSR    Introduction Nitrogen is the major limiting nutrient for most plant species (Green wood, 1982). Plants require nitrogen from soil or from atmosphere by symbiotic nitrogen fixation (Vance, 1990). Legume capable of fixing atmospheric nitrogen in symbiotic association with Rhizobium improves soil nitrogen content in uncultivated land (Sanginsg et. al. 1988). Inoculation of legume crop with Rhizobium strain having better symbiotic crop productivity (Singh & Singh 1983a, 1983b). Antibiotic resistance is found in antibiotic producing microorganisms which need the mechanism for self protection. (Gray & fitch 1983: Trieo-cuot et al 1987a). Cole & Elkan 1973,1979 repotted that resistances of antibiotics were plasmid born. The core replicons of self transmissible plasmid involved in antibiotics resistance gene transfer were devoid of resistance determinants in the bacteria (Hughes & Datta, 1983). The bacteria possess a differential response towards the antibiotics as some show resistance & some susceptibility for a particular antibiotic. Marker antibiotics are evaluated as confirmatory test of their identity as well as control. (Prasuna, 1987). The present investigation has been undertaken to study antibiotic resistance towards different Rhizobium spp.  Material and Methods Rhizobia were isolated from root nodules of 10 cultivated viz – Phaseolus aureus (PA-R1), Phaselus vulgaris (PV-R2), Arachis hypogaea (AH-R3). Dolichos lablab (DL-R4) Glycine max (GM-R5), Trigonella foenum graecum (TFg-R6). Cicer arietinum (CA-R7), Vigna ungiculeta (VU-R8), Pisum sativum (PS-R9). Lathyrus sativa (LS-R10) & 3 wild plants viz : - Mimosa pudica (MP-R11),  Desmodium triflorum (DT-R12), and Tephrosia purpurea (TP-R13), Isolations were made from nodules by standard procedures taking only healthy nodule from root of each plant (Vincent 1970). Nodules were selected & surface sterilized with 0.1% (w/v) mercuric chloride (Hgcl2)  for one minute & washed thoroughly several time with glass distilled water (Sloger 1969). Pure Rhizobia isolates were transferred on yeast extract mannitol agar (YEMA) slant. Susceptibility test was performed by using the paper disc diffusion assay. Five antibiotics were used viz – streptomycin, Penicillin-G, Erythromycin, Teramycin & Norfloxacin. The biodiscs were prepared in three different concentration viz : 0.5%, 1.0% & 1.5%. Bacterial suspensions were prepared in nutrient broth medium. Each test tube containing 10 ml medium & one loop full Rhizobia were inoculated. One ml of bacterial suspension & 10 ml of nutrient agar medium were inoculated in petriplate. After plating & solidification of the medium biodiscs were placed upon the surface of solidified nutrient agar medium. The plates were incubated for 24 hrs. at 28±1°C . The zone formation was measured in mm (Prasuna, 1987). Result and Discussion  The study of antibiotics sensitivity tests showed that amongst cultivated hosts streptomycin was sensitive in Rhizobium sp. Isolated from R6 (25 mm) followed by R7, R2 = R5, R1, R4, R8, R3, R9. Streptomycin could not inhibit the growth of Rhizobium spp. Isolated from R10 (No zone formation). The inhibition zone indicate sensitivity of different isolates. R6 was susceptible & R10 showed resistance towards streptomycin. Sensitivity can be expressed as R6> R7> R2= R5> R1> 4> R8> R3> R9> R10. Penicillium was highly sensitive towards R5 (39 mm) followed by R7, R1, R6= R3, R10, R8, R2, was least sensitive towards R4 (0.5 mm) & R9 was resistance towards Penicillin (No zone Formation). The sequence of sensitivity can be expressed as R5 > R7> R1> R6 = R3> R10> R8> R2> R4> R9. Teramycin was highly sensitive towards R3 (35mm) followed by R1, R10, R5, R9, R4, R3, R7, R6, Teramycin could not inhibit the growth of Rhizobium spp isolated from R8. Response of Teramycin was in the order of R2> R1> R10> R5> R9> R4> R3> R7> R6> R8. Erythromycin was highly sensitive towards R3 (26 mm) followed by R7, R6 = R2, R9, R10= R5, R1, R8 - Erythromycin could not inhibit the growth of R4. Sensitivity can be expressed as R3> R7> R6 = R2> R9> R10= R5> R1> R8> R4. Norfloxacin was highly sensitive towards R7 followed by R9 = R1, R6, R3, R4, = R5, R5; R10 & was least sensitive towards R8 (22mm).S. Sharma and  R. Diwan/J Ecobiotechnol 3 (2011) 13-15     14  Table 1 : Effect of  Antibiotics on the growth of  Rhizobium  Species Name of Antibiotics Concentration Name of legume hosts Cultivated Wild % PA PV Tfg DL GM AH PS LO CA VU DT MP TP (1)Streptomycin 0.5 18 17 17 14 19 12 10    - 15 12 14    - 21 1 19 19 23 18 21 17 11    - 20 17 16 0.8 23 1.5 22 23 25 21 23 19 17    - 24 20 17 1.2 28 (2)Penicillin 0.5 22 10 23    - 31 19  - 11 28 12 13 8    - 1 24 13 25 0.4 34 23  - 17 29 15 16 10  - 1.5 24 14 26 0.5 39 26    - 22 32 17 19 13  - (3)Teramycin 0.5 28 20   - 17 17 18 16 18 16   -   15 11 15 1 30 28 1 18 18 19 19 20 18  - 17 14 18 1.5 31 35 1 21 23 20 22 25 19  - 19 16 21 (4)Erythromycin 0.5 11 10 18  -  10 22 16 11 17 3 20 16 6 1 12 13 19  - 10 25 16 12 20 4 22 20 11 1.5 14 22 22 - 15 26 17 15 23 4 25 21 14 (5)Norfloxacin 0.5 25 25 26 26 24 29 26 21 30 19 25 26 20 1 29 27 28 27 25 30 30 23 32 20 28 27 24 1.5 32 28 31 28 28 30 32 27 35 22 30 28 28                                       S. Sharma and  R. Diwan/J Ecobiotechnol 3 (2011) 13-15     15                         1- Effect of antibiotics on the growth of different Rhizobium spp  However zone formation of R4, R5 & R2 was equal. Respons of norfloxacin was in the order of R7> R9 = R1> R6> R3> R4= R5= R2> R10> R8.  In wild Legume hosts streptomycin & Teramycin weres highly sensitive in R13 followed by R12 & was least sensitive in R11. In Penicillin, Erythromycin & norfloxcin was highly sensitive in R12 followed by R11 & was least sensitive in R13. However penicillin could not inhibit the growth of Rhizobium spp. Isolated from R13 (No zone formation) & zone formation of R13 & R11 was equally found in Norfloxacin. The finding of the present study revealed that norfloxacin is more effective followed by Penicillin, Teramycin, Erythromycin & Streptomycin. Norfloxacin is a flurequinolence antibacterial agent effective against several Gram negative bacteria (Kamath et al. 1992). The bacteria possess a differential response towards the antibiotics as some show resistance & some susceptibility for a particular antibiotic. Any sign of growth inhibilion was scored as sensitivity to that antibiotic. which means that “resistance” was very striclly defined so that no organism with any sign of sensitivity would be classified as resistant. References Cole, M.A. & Elkan, G.H.  (1973): Transmissible resistance to penicillin G, neomycin & chloramphenicol in Rhizobium japonicum. Antimicrobial Agents & Chemofhempy. 4, 248-253. Cole,  M.A.  &  Elkan,  G.H.  (1979)  :  Multiple  antibiotic resistance in Rhizobium japonicum. Applied & Environmental microbiology, 37,867-870. Gray, G.S. & Fitch, W.M. (1983) Evolution of antibiotic resistance genes  :  the DNA sequence of a kanamycin resistance gene from staphylococcus aureus. Molecular Biology & Evolution, 1, 57-66. Green wood D.J. (1982) Nitrogen supply & Crop yield : The Global Scene : Plant soil : 67, 45-49. Hughes, V. M. & Datta, N. (1983) : Conjugative plasmids in bacteria of the "pre antibiotic" era, Nature, 302, 725-726. Kamath, R.U. Singh, U.V. & Udupa. N. (1992) : Indian J Pharm Sci., 4,148. J7'.  Prasuna,  P.L.  (198?')   :  Legume  Rhizobium  nitrogen  fixing Symbiosis.    Ph.D.    Thesis    Ravishankar University, Raipur (C. G.). Sanginga N. Mulongoy K & Ayanoba A (1988) Response of leucaena/Rhizobium  symbiosis  to  mineral nutrients in south western Nigeria plant & boil 112 : 121-127. Singh, B.P. & Singh, H.G. (1983a) comparative efficacy of S on  production  of  green  matter,  mineral composition & uptake of mustard on vertisols. -. Forage Research. 9:37-41. Singh, B.P. & Singh, H.G. (1983b) Inter relationship between soil & foliar applied sulphur on oil percent in seed & oil yield of mustard Crop Physiology. 1:63-76. SIoger C, (1969) Plant Physical, 44 : 1966. Subba Rao, N.S. (1988) : Biofertilizers in Agriculture. Oxford & IBH Pub.Co.Pvt.Ltd., New Delhi. PP. 248. Trieu-Cuot, P. Arthur, M. & courvalin, P. (1987) Origin, evolution   &   dissemination   of   antibiotic resistance  genes.  Microbiological sciences, 4, 263-266. Vance, C.P. (1990) Symbiotic nitrogen fixation : recent genetic advance. In The Biochemistry of Plants : Intermediary Nitrogen Metabolism Mifin B. J. Lea P.J. eds. vol. 16 pp. 48-88, Academic press, Sen Diego. Vincent, J.M.  (1970)  : "A Manual for the Practical Study of Root  Nodule  Bacteria"  Blackwell  Scientific Publication, Oxford&Edinburg.   

Characterisation of Rhizobia on the Basis of Antibiotic Responses

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Characterisation of Rhizobia on the Basis of Antibiotic Responses

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