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Evaluating the potential of Rhizobium Rhizogenese in production ouxin and nitrogen fixation.


Rhizobacteria are the bacteria that their ecological nest are in the soil beyond of roots or root surface, where bacteria can grow and multiply at different stages of plant growth. Phrase of PGPR has been applied for a variety of bacteria that had significant effect on plant growth [1]. Rhizobium rhizogenese is a gram-negative, aerobic and a soil born bacterium which is belong to family rhizobiacea with a wide distribution in the world. Agrobacterium was described by Conn in 1942, and then was placed in rhizobiacea family by Yang et al based on the sequence of 16S rRNA, and then was called Rhizobium rhizogenese [2]. Stimulate plant growth by PGPR bacteria is done by variety ways, such as fixing molecular nitrogen, production of plant growth-stimulating hormones, manufacture vitamins and increase nutrient elements solubility. Two main features of bacteria are hormone secretion and plant growth regulators. Among the plant hormones, auxin and ethylene have crucial role in the development of plant's root system, increase the absorption of water and nutrients and as a consequence increase the yield [1]. One of the most important ways that R. rhizogenese affects on plants by synthesis of indole phytohormone (IAA) that is absorbed by the root system that this hormone causes development of roots and as a result increase of plant nutrient uptake. Many researchers have studied on effects of R. rhizogenese on root growth. Sharma et a [l5]. Showed that the strains of R. rhizogenese were isolated from the roots of wheat produced the highest levels of indole acetic acid and after inoculation, that isolates increased to 61.3% and 26.6% in root divisions and root length respectively Compared to the control [3]. Nitrogen is one of the macronutrients for plants, which is present in small amounts in the soil. 80% of the volume of air is nitrogen gas, but this form of nitrogen is not usable by plants because plants only could absorb nitrogen in forms of nitrate and ammonium. There are some microorganisms that are capable to convert molecular nitrogen to ammonium, they are called nitrogen fixation bacteria and all of these organisms are prokaryotes. Nitrogenase is a bacterial enzyme that is responsible for reduction of N2 to NH3 and can lead the reactions in normal pressure and temperature. This kind of prokaryotes is so-called diazotroph, that their required energy obtain through solar energy or chemical oxidation [4]. Stephens et al showed that soybean plants inoculated with R. rhizogenese were contain of nitrogen through biological fixation had significantly greater than control [5]. Friend et al showed that the isolate IRBG74 of the species R. rhizogenese addition to nitrogen fixation in root nodules of soybean is also a diazotroph of stimulate plant growth [6]. Baset mia and Shamsuddin reported that R. rhizogenese is a free-living nitrogen fixing and producer IAA and hairy roots in the host plant and also the bacteria can increased plant performance like Rhizobium [7]. One of the main criteria to evaluate the efficiency of growth promoting bacteria is molecular nitrogen fixation and auxin production therefore, the present study examined the ability of local isolates of R. rhizogenese as a PGPR bacteria.


28 samples of soybean plant roots with the soil of around it were collected from 28 spot of the Golestan province. Then in order to isolate R. rhizogenese of the soybean plants, aerial part were cut off and nodes on the root were isolated. For sterilization first nodes were placed in alcohol for 30 seconds and then in 1% sodium hypochlorite for 5 minutes at the end were rinsed several times with distilled water [2], 1 to 2 large root nodules of each sample were selected and placed in the ordinary test tubes containing 1 ml of sterile distilled water and were crushed by the pestle, Then 100 ml of sterile nodes extract were transferred by pipette to YMA medium and were spread in surface, then cultured plates were placed for 48 h in a growth chamber. To compare the shape and morphological characteristics of foreign isolates of R. rhizogenese GMI, which had been prepared from the Institute of Biotechnology in Tehran, was cultured on YMA medium. Rhizobium strains with similar characteristics to the foreign isolate (convex, slimy or round) to perform additional tests for more identification were isolated and purified. identification tests include: 1. gram Test, 2. Catalase Test 3. sodium chloride tolerance, 4. Congo red test, 5. growth on potato dextrose agar (PDA), 6. Growth in Hofer's alkaline broth, 7. Growth in Glucose Peptone Agar, 8. Growth and pigmentation in ferric ammonium citrate broth, 9. 3-ketolactose test, 10. R. rhizogenese produced yellow ring of precipitate of CuO around the colonies of the bacterium in lactose agar media, 11. Hairy root formation test [2].

Auxin production test:

Measurement of auxin production were done by using spectrophotometrical method with salkowski reagent. For this purpose, bacteria were cultured on YMA medium and were kept for 3 days at 28[degrees]C. The grown colonies were inoculated to the flasks containing 20 ml of LB medium with concentration 100 mg L-Tryptophane [8].The mentioned flask then were centrifuged after 72 hours (Model SIGMA) (10 minutes during 10,000 rpm), the mixture of sample and reagent Salkowsky (2 ml half mM Fe[Cl.sub.3] and 98 ml 35% HClO4) was prepared as a 2:1 ratio. The samples were subjected in laboratory temperature for 20 minutes. With a spectrophotometer (model BRITE) light absorption at a wavelength of 530 nm was read. The amount of auxin produced by comparing the absorbance (OD) with a standard prepared curve from indole acetic acid (IAA) was calculated. For drawing auxin standard curve, concentrations of 0, 10, 20, 30, 40, 50, 60, 80 and 100 mg per L IAA and one ml of this solution was well mixed with two ml of reagent Salkowsky. After 20 min at a wavelength of 530 nm was measured [8].

Molecular nitrogen fixation test:

To investigate the molecular nitrogen fixation, method of acetylene reduction (ARA) and gas chromatography (GC) was used. First within 13 mm test tubes containing 5 ml of free N YMA medium was poured into sterile environment. Then the test tubes were inclined to increase the level of bacteria were cultured for, After perfect cooling of environment achieve to the desired temperature to grow and 100 ml of a 48-hour suspension bacteria was inoculated into culture medium. Then the medium were inoculated with bacteria into the growth chamber at 28 [degrees] C for 48 h. After 48 hours incubation, the tubes with cotton plugs were replaced with sterile plastic doors and 10% of the volume of air inside the tube (0.7 ml including the volume of rubber) were discharged by syringe and same amount of acetylene gas was injected to the tubes, seams were covered with teflon rubber stopper. After 24 hours of incubation 0.7 ml air of the tubes was injected into the gas chromatograph (model GOW_MAC), using a Hamilton syringe and the area under the generated curve was read. However, before reading the bacterial samples, the instrument was calibrated with different concentrations of ethylene and calibration curves were plotted [9].


Based on 11 identified test, 10 isolates attributed to R. rhizogenese were isolated, the results showed isolates all were: Gram-negative, catalase-positive, capable of growth on YMB medium containing 3% NaCl, were capable of absorbing Congo red in medium with this test R. rhizogenese were isolated from Rhizobium, Rhizobium are not be able to absorb Congo red, isolates capable of growth on PDA medium and able to grow in the Hofer's alkaline broth at PH = 11, with doing this test, R. rhizogenese was isolated from Rhizobium and bradyRhizobium. Strains were able to grow on glucose peptone agar, grow and pigmentation in ferric ammonium citrate media, and were able to use 3 - ketolactose instead of mannitol in the culture medium. Strains able to grow on lactose agar and were formed a yellow precipitate around colonies after using Benedict's reagent, with this test R. rhizogenese were isolated from other species of Agrobacterium. Finally strains were able to establish hairy root on carrot sterilized discs that were placed on MS medium (figure 1).

Results of production of axuin test:

Quantitative test of the ability to produce auxin hormone (IAA) showed that strains of R. rhizogenes were able to produce the auxin hormone (IAA) in medium containing tryptophan and this ability was different among R. rhizogenes strains. According to the figure 1 the 10RR isolate with 93.62 mg per liter and the 22RR isolate with 63.42 mg per liter produced the highest and lowest IAA respectively.

3.2 Ability of molecular nitrogen fixation:

In the present study, nitrogenase activity of 10 isolates were examined on native R. rhizogenese isolates. The results showed that all isolates were able to fix molecular nitrogen or in other words the reduction of acetylene to ethylene was measurable by GC. Figure 2 showed that strains were capable of producing an average 45 nmol ethylene per tubes on time. The isolates 5R.R and 18R.R with producing 62.4 and 43.6 nmol ethylene h pipe, were accounted for the highest and lowest ethylene production respectively.


Different components of plant growth promoting (PGPR) strains showed that native R. rhizogenese had the ability of production auxin and nitrogen fixation. Auxin production and nitrogen fixation in most of the isolates, were R.R10 and R.R5 respectively. We can conclude that R. rhizogenese the main performance is in genetic engineering and gene transfer can also be used as a plant growth promoting rhizosphere bacteria.


Article history:

Received 15 Feb 2014

Received in revised form 24

February 2014

Accepted 15 April 2014

Available online 5 May 2014


I am really thankful to my teachers and Gorgan university, Golestan, Iran for providing laboratory facilities.


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(1) Maryam Sarayloo, (2) Mohsen Olamaee, (3) Mohammad Hosein Arzanesh

(1) Soil Biology M.Sc Student, Gorgan University of Agricultural Sciences and Natural Resources, Gorgan, IRAN

(2) Professor Assistant, Department of Soil Science, Gorgan University of Agricultural Sciences and Natural Resources, Gorgan, IRAN.

(3) Professor Assistant, Agricultural and Natural Resources Research Center of Golestan, Gorgan, IRAN.

Corresponding Author: Maryam Sarayloo, Soil Biology M.Sc Student, Gorgan University of Agricultural Sciences and Natural Resources, Gorgan, IRAN

Table 1: Analysis of variance of ethylene and auxin
production by the isolates.

Mean squares

Source of      freedom    ethylen     auxin
variation      degree

Bacteria          10      76.489 **   477.0794 **
error             22        0.682        9.206
total             32
cv                          1.77         4.29

** Significant at 1% level [R.sup.2] = 98%
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Author:Sarayloo, Maryam; Olamaee, Mohsen; Arzanesh, Mohammad Hosein
Publication:Advances in Environmental Biology
Article Type:Author abstract
Date:Feb 14, 2014
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