Research Article

Studies on biosynthesis of auxin in rhizobium and their impact on growth of Vigna mungo L.

Parthiban P.1, Shijila Rani A.S.1, Mahesh V.2, Ambikapathy V.3*

1Department of Microbiology, Marudupandiyar College, Vallam, Thanjavur, Tamil Nadu, India

2MR Arts and Science College, Mannargudi, Tamilnadu, India

3Department of Botany and Microbiology, AVVM Sri Puspham College, Poondi, Tamil Nadu, India

*For correspondence

Ambikapathy V.,

Department of Botany and Microbiology, AVVM Sri Puspham College, Poondi, Tamil Nadu, India.

Email: drva1967@gmail.com

 

 

 

 

 

 

Received: 21 April 2016

Revised: 02 May 2016

Accepted: 11 May 2016

ABSTRACT

Objective: The present investigation, the use of plant growth promoting rhizobacteria was used to growth and improves the yield in crop of Vigna mungo.

Methods: Rhizobium strains were isolate and identified by Yeast Extract Mannitol Agar medium with congored (CYEMA) and some biochemical test were performed. Separation and quantification of growth hormone by paper chromatography and spectrophotometric methods followed and its impact of Vigna mungo in green house experiments.

Results: The Rhizobium strains were isolated from legumes plant Mimosa pudica L, Arachis hpogea L and Vigna mungo L. Indole-3-acetic acid (IAA) produced from the L-trptophan medium and identified by using paper chromatography techniques. The pot culture experiments were conducted with various treatment of Rhizobium sp, strains are high growth and yield.

Conclusions: Rhizobium is beneficial organisms and to interaction with growth promotion to all leguminous plants as well as nitrogen fixation, its biological process of the ecosystem. It is concluded to the Rhizobium species is eco-friendly to farmer and these environments.

Keywords: Root nodules, Rhizobium, IAA, Vigna mungo L. seeds

Introduction

Growth hormones are substance synthesized in particular cells, in extremely small quantitive influence developmental process. Plant growth hormones was vital role in controlling plant growth such as, Auxins, gibberllins, cytokines, ethylene, and abiscissic acid, they are naturally synthesized by some microbes. Bacteria are abundantly present in the rhizosphere and close in vicinity of the root. It has been recongnized that several generea of these rhizobacteria have the ability to promote plant growth and these have been termed plant growth promoting rhizobacteria or PGPR.1

Rhizobia are well known for their capacity to establish a symbiosis with legumes. The interaction between Rhizobium bacteria and leguminous plants results in nodule formation and atmospheric nitrogen is converted to ammonia and then metabolized by the plant2. They also produce plant hormones like auxins, gibberellins cytokinines, ethylene, and abscisic acid in addition to symbiotic nitrogen fixation. Among the phytohormones, auxin was the first plant hormone discovered.2 Several workers reported that Rhizobium sp. produced high amount of auxin in culture with or without L-tryptophan in the YEMA medium.

The phyto hormones auxin is synthesized in the shoot tips of growing plants. The growth hormone auxin was elongation growth, cell division and differentiation and apical dominance. Zinc is essential element for auxin biosynthesis. The highest concentrations of auxin were found in growing tips, buds, leaves and roots of the plants.

Materials and Methods

Sample collection: Root nodules were collected from young and healthy seedling plants of Mimosa pudica L, Arachis hpogea L and Vigna mungo L plant from Oruthanadu, Thanjavur (dist), Tamil nadu, India.

Isolation of Rhizobium sp from root nodules: To select, healthy and reddish-pink color root nodules were tenderly washed with distilled water and surface sterilized by keeping in 0.1 percent HgCl2 for 4-5 min and wash with sterile distilled water followed by 95 percent ethyl alcohol, repeat washing with sterile distilled water.3 The nodules were crushed in a small drop of sterile distilled water with the help of sterile glass rod and obtain milky suspension. Then, serial dilutions were made and aliquots dilutions were spread on yeast extract mannitol agar (YEMA) medium and plates were incubated at 28±1°C.4

Identification and biochemical test of Rhizobium sp: The isolated colonies were confirmed by microscopic observation and biochemical test6,7 and cultural test were performed. Isolates were identified as per Bergey's manual of Systematic bacteriology.

Biosynthesis of IAA: Then, the colonies were grown individually in trypticase soya broth (TSB) for 30 min, and one ml of cell free culture filtrate to each strain was added to two ml of Salkowaski reagent and incubated at 28±2°C for 48 hours in shaking incubator at 1000 rpm. The content was filtered through Whatmann No: 1 filter paper before measuring IAA in terms the broth, these contents was determined by colorimetrically at 530 nm.8

Identification of IAA: Biosynthesized auxins were partially characterized by paper chromatography method.9 In which three, grams of culture filtrate were extracted with 50 ml of peroxide-free ether for 2 hours at 5oC. The appearance of the strips under ultraviolet light and spraying with modified Salkowski reagent is indicated.

Pot culture method: The pot trails were accomplished by Department of Microbiology Marudupandiyar College, Vallam, Thanjavur. Each one had conducted triplicates. The treatment follows as:

T1 Control (without Rhizobium); T2 Rhizobium (R1 seed inoculation); T3 Rhizobium (R2 seed inoculation); T4 Rhizobium (R3 seed inoculation); T5 Rhizobium (R1 seed inoculation +foliar spray); T6 Rhizobium (R2 seed inoculation + foliar spray); T7 Rhizobium (R3 seed inoculation + foliar spray).

After germination, plants were treated with 48 hrs old culture, which were foliar sprayed to the Vigna mungo plants by every week, starting from 10th day of germination. The plants were harvested every 15th day of intervals analyze morphometric, biometric and number of root nodules were analyzed.

Estimation of carbohydrates10: Fresh leaves 100 mg was taken in a test tube and hydrolysed with 2 ml of concentrated H2SO4 for 30 minutes at 100°C. To 0.5 ml of 5 percent phenol and 5 ml of conc. H2SO4 were added and mixed thoroughly. The colour developed was measured at 490 nm in Spectronic 20. Glucose was used as a standard.

Estimation of total proteins11: 100 mg of fresh leaves was ground in mortar and pestle with 1.0 ml of phosphate buffer and centrifuged at 10,000x g for 10 minutes. To 0.5 ml and 5 ml of alkaline copper reagent were added and allowed to stand for 10 minutes, finally 0.5 ml Folin's reagent was added. The absorbance was measured after 30 minutes at 750 nm in spectronic 20. The standard graph was prepared by using Bovin Serum Albumin.

Results and Discussion

In the present investigation, the isolates of rhizobia from root nodules of Arachis hypogea and Vigna mungo plants were characterized by opaque and milky white in appearance in YEMA medium. The isolated rhizobial strains were identified in microscopic and biochemical test to perceive as positive results (Table 1). All strains were gram negative and did not absorb red color when cultured in YEMA containing congo red. The result were compared with Bergey's manual of Determinative Bacteriology and conformed as Rhizobium. These Rhizobial isolates produced auxins in-vitro conditions in the ranges from 3.00 to 3.8 mg/L, IAA equivalents without L-tryptophan. The pot trial was conducted to study the response of Vigna mungo to inoculation with rhizobium and foliar application. The result revealed that the black gram responded well to the inoculation of rhizobium. The plants inoculated with native rhizobium possessed significantly greater shoot, root height, dry weight, nodule numbers, total carbohydrate and protein and nodules formation was monitor and tabulated (Table 3, 4, Figure 1 and 2).

Table 1: Microscopic and biochemical characterization of Rhizobial isolates.

S. No

Characteristics

Results

1

Gram staining

-

2

Motility

+

3

Indole test

+

4

Methyl red test

-

5

Voges-Proskauer test

-

6

Citrate utilization

+

7

Starch hydrolysis

-

8

Urea hydrolysis

+

9

H2S production

-

10

Catalase test

+

11

Lactose

-

12

Maltose

+

Table 2: Quantitative assessment of growth hormone production.

S.No

Treatment

IAA(mg/ml)

1

R1

3.00

2

R2

3.20

3

R3

2.02

Figure 1: Effect of rhizobia on carbohydrate content of Vigna mungo.

Figure 2: Effect of rhizobia on protein content of Vigna mungo.

Shahzad et al.12 isolated Rhizobium from root nodules of Alfalfa (Medico sativa) plant and characterized on the basis of various biochemical tests. Previously, Ashfaq et al.13 the rhizobial strains were isolated from the nodules of Mung bean and Pisum sativum.14 Similarly,
Singh et al.15 also characterized Rhizobium strains on the basis of biochemical tests. Rhizobium is symbiotic bacteria which form nodule in leguminous plant. Auxin production is frequently pointed out.13,16-20 Different legume nodulating rhizobial strains preferred different vitamins sources for IAA production reported.21,22 The current observations of the pot experiment of pea was close to Fischer et al.23 who find out the ability of Rhizobium on wheat by development of shoot/ root fresh and dry weights. All the Rhizobium strains improved the root and shoot dry biomass by 100% and 70% respectively. Some workers reported in who obtained 70% increase in pea root/shoot dry biomass by PGPRs inoculation as contrast to control (uninoculated).24

Table 3: Effect of Rhizobium on plant height and biomass of Vigna mungo from 15th days of treated with inoculums.

S. No

Treatment

Plant height (cm)

Plant dry weight mg/plants

No. of nodules

shoot

root

1

T1

16.5

4.5

85.0

-

2

T2

20.5

5.1

139.0

-

3

T3

20.5

6.7

137.0

-

4

T4

20.5

5.5

124.7

-

5

T5

20.3

6.5

123.7

-

6

T6

23.3

5.5

83.0

-

7

T7

20.3

5.7

125.4

-

T1 Control (without Rhizobium); T2. Rhizobium (R1 seed inoculation); T3 Rhizobium (R2 seed inoculation); T4 Rhizobium (R3 seed inoculation); T5 Rhizobium (R1 seed inoculation +foliar spray); T6 Rhizobium (R2 seed inoculation + foliar spray); T7 Rhizobium (R3 seed inoculation + foliar spray)

Table 4: Effect of Rhizobia on plant height and biomass of Vigna mungo after 30 day treatment.

S. No

Treatment

Plant height (cm)

Plant dry weight mg/plants

No. of nodules

shoot

root

1

T1

18

6.4

89.0

-

2

T2

22

8

145.0

3

3

T3

22.5

7.2

143.0

3

4

T4

22

7

130.7

4.2

5

T5

23

10

131.0

-

6

T6

25

8

92.0

-

7

T7

19

17

130.4

-

T1 Control (without Rhizobium); T2. Rhizobium (R1 seed inoculation); T3 Rhizobium (R2 seed inoculation); T4 Rhizobium (R3 seed inoculation); T5 Rhizobium (R1 seed inoculation +foliar spray); T6 Rhizobium (R2 seed inoculation + foliar spray); T7 Rhizobium (R3 seed inoculation + foliar spray)

Conclusions

From this study, finish off that the rhizobia was the most common plant growth promoting bacteria to produce plant growth promoting hormones of auxin. Arachis hypogia isolate can be promoting early blackgram growth, besides increasing plant height, dry weight, total carbohydrate and protein. However, further studies are needed for an integrative understanding of hormones and metabolism in Rhizobium.

Acknowledgements

The authors wish to thankful of Mardupandiyar College, Vallam, Thanjavur, for extending the facilities to carry out the laboratory studies.

Funding: No funding sources

Conflict of interest: None declared

References

  1. Glick BR, Patten CL, Holguin G, Penrose DM. Biochemical and genetic mechanisms used by plant growth promoting bacteria. Imperial College Press, London; 1999.
  2. Vincent JM. Factors controlling the legume Rhizobium symbiosis. In WE Newton, WH Orme-Johnson, eds, Nitrogen Fixation 11. University Park Press, Baltimore, MD; 1980: 103-109.
  3. Darwin CR. The power of movement in plants London, Murray; 1880.
  4. Russel AD, Hugo WB, Ayliffo GAJ. Principles and practices of disinfection, preservation and sterilization. Black wall scientific, London; 1982.
  5. Somasegaram P, Hoben MJ. Hand book for Rhizobia: methods in legumen. Rhizobium technology, New York. Springer-verlag, Hawaii, NIFTAL; 1985: 450.
  6. Josey DP, Beynon JL, Johnston AWB, Beringer JE. Strain identification in Rhizobium using intrinsic antibiotic resistance. J Appl Bacteriol. 1979;46:343-50.
  7. Gordon SS, Weber RP. Colorimetric estimation of indoleacetic acid. Plant physiology. 1951;26:192-5.
  8. Mostafa SM, Ghazi SM, George NM. Effect of foliar spraying with alar on the endogenous growth regulation substance of water melon fruits. Annals Agric Sci Fac Agric. 1990;35(1):15-29.
  9. Dubois M, Gilles RA, Hamilton FK, Roberts PA, Smith F. Colorimetric methods for determination of sugar and related substance. Anal Chem. 1956;28:350-6.
  10. Lowry OH, Rosebrough NJ, Farr AL, Randal RJ. Protein measurement with the folin phenol reagent. J Biol Chem. 1951;193:265-75.
  11. Shahzad F, Shafee M, Abbas F, Babar S, Tariq MM, Ahmad Z. Isolation and biochemical characterization of Rhizobium meliloti from root nodules of Alfalfa (Medico sativa). J Animal Plant Sci. 2012;22(2):522-4.
  12. Ashfaq M, Rehman MA, Ali A. The impact of optimum dosages of mineral in various combinations on larval development and silk production of Bombx mori L. Park J Biol Sci. 2000;3:1391-2.
  13. Deshwal VK, Chaubey A. Isolation and Characterization of Rhizobium leguminosarum from Root nodule of Pisum sativum L. Journal of Academia and Industrial Research. 2014;2(8):464-7.
  14. Singh B, Kaur R, Singh K. Characterization of Rhizobium strain isolated from the roots of Trigonella foenumgraecum (fenugreek). Afr J Biotechnol. 2008;7(20):3671-76.
  15. Biswas JC, Ladha LK, Dazzo FB. Rhizobia inoculation improves nutrient uptake and growth of lowland rice. Journal of Soil Science. 2000;64:1644-50.
  16. Khalid A, Arshad M, Zahir MA. Screening plant growth promoting rhizobacteria for improving growth and yield of wheat. Journal of Applied Micrbiology. 2004;96(3):473-80.
  17. Chi F, Shen SH, Cheng HP, Jing YX, Yanni YG. Ascending migration of endophytic rhizobia, from roots to leaves, inside rice plants and assessment of benefits to rice growth physiology. Applied Environ Microbiol. 2005;71:7271-8.
  18. Lee HS, Madhaiyan M, Kim CW, Choi SJ, Chung KV, Sa TM. Physiological enhancement of early growth of rice seedlings (Oryzae sativa L.) by production of phytohormones of N2 fixing metholotrphic isolates. Biol Fert Soils. 2006;42:402-8.
  19. Ambika R, Kavitha P, Panneer Selvam A, Sengottaian N. Production and extraction of Indole acetic acid by using efficient strain of Rhizobium isolated from maize. World J Pharm Sci. 2014;2(4):294-7.
  20. Ghosh PKJ, Ganguly P, Maji T, Maiti TK. Production and composition of extracellular polysaccharide synthesized by Rhizobium undicola isolated from aquatic legume, Neptunia oleracea Lour," Proceedings of the National Academy of Sciences, India Section B: Biological Sciences; 2014.
  21. Ghosh PKJ, Kumar De T, Kanti Maiti T. Production and Metabolism of Indole Acetic Acid in Root Nodules and Symbiont (Rhizobium undicola) Isolated from Root Nodule of Aquatic Medicinal Legume Neptunia oleracea Lour. Journal of Botany. 2015;24(5):35-46.
  22. Fischer SE, Fischer SI, Magris S, Mori GB. Isolation and characterization of bacteria from the rhizosphere of wheat. World J Microbiol Biotechnol. 2007;23:895-903.
  23. Hilali A, Prevost D, Broughton WJ, Antoun H. Effect of inoculation with strains of PGPR on the Wheat in Morocco soils. Crop Sciences 2001;47(6):590-3.
  24. Ahmad F, Ahmad I, Khan MS. Screening of free-living rhizospheric bacteria for their multiple plant growth promoting activities. Microbiology Research. 2008;163:173-81.

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