Isolation and Diagnosis of the Root Nodule Bacteria Associated with Mung Bean Plant in Gypsiferous Soil and Testing the Promotional Criterion of Isolates

The root nodule bacteria are utilized in the production of natural biological fertilizers to achieve clean agriculture by reducing chemical fertilizers. In this study, 35 samples of the root nodules associated with mung bean plants were collected from various agricultural areas of Iraq, Salah Al-Din Governorate on 4\7\2022, ten samples isolated by growing them on yeast extract mannitol agar (YEMA) and the phenotypically pure isolates were diagnosed based on the culture, microscopic and biochemical characteristics., The phenotypic diagnosis results showed that The color of the colonies was between white, creamy and yellow, Spherical, convex and smooth, gram-negative, was able to move and pink and light pink on Congo red stain medium and 9 isolates it was fast growing as it gave yellow color on YEMA-BTB bromothymol blue medium except for one isolate it was slow growing as it gave blue color and all isolates unable to grow on Hofer alkaline medium except for one isolate, The efficient isolate was selected in the production of indole acetic acid, chelating compounds and phosphate solubilization,. The results showed that isolates (M3, M4, M5, M6, M7) have a high ability for iron chelation. It was found that the (M6) isolate gave the highest phosphate solubilization and indole production, which showed a phosphorus solubilization of 39.504 mg P.L-1 and an indole production rate of 21.5 μg.ml-1. This was followed by the isolate (M3), which showed a phosphorus solubilization of 23.723 mg P.L-1 and an indole production rate of 17.2 μ.g.ml-1. Molecular diagnosis was performed for five competent isolates in the production of Indole acetic acid, production of chelating compounds, and phosphate solubilization. The isolates were molecularly diagnosed by PCR, the 16SrRNA gene was amplified, then the sequence of nitrogenous bases was analyzed and when matching with the global strains included in the NCBI Genetic Bank website, The results of molecular diagnosis showed that two isolates belong to Rhizobium bacteria. the results showed that (M3) isolate is %99.50 similar to the Rhizobium leguminosarum isolate OTU21_I. and the results showed that (M6) isolate is 99.48% similar to the Bradyrhizobium japonicum strain A3 and 99.96% to the Bradyrhizobium japonicum, strain:NK5 and therefore the isolates is genetically close to Rhizobium leguminosarum strain AE15 and Bradyrhizobium japonicum strain AE14 bacteria and has been recorded in the Global genome bank under accession number OP975690 and version number OP975690.1 for Rhizobium leguminosarum and accession number OP975688 and version number OP975688.1 for Bradyrhizobium japonicum and this record is the first for this bacteria which associated with mung bean in gypsiferous soils in Iraq.


Introduction
There are many problems associated with gypsiferous soil and related to plant nutrition, including the imbalance of nutrient ions resulting from saturation of the soil solution with sulfate and calcium ions, and their effects on root growth and spread, as well as the low content of clay and organic matter.Therefore, recent research has focused on using complementary methods to increase the availability of nutrients in the soil, such as using bio-fertilizers to reduce pollution problems, which are natural and inexpensive sources of plant nutrition compared to chemical fertilizers in nutrient-poor soils [1].Microorganisms are used to improve agricultural productivity, as well as the quality of crops [2,3].Rhizobia bacteria are microorganisms capable of nitrogen fixation through a symbiotic relationship with the legume plant family.They fix approximately 65% of the nitrogen used in sustainable agricultural production of legume crops ]4[.The most important genus capable of symbiotic nitrogen fixation is Rhizobia, which is characterized by its ability to form nodules on the roots of leguminous plants.Rhizobia bacteria in leguminous plants provide 70-80% of the plant's nitrogen requirements [5].Mung bean (Vigna radiata L), which belongs to the Leguminosae family, is one of the most important legume crops in the world, and is one of the most common crops in most tropical and subtropical regions [6].Mung bean was originally cultivated mainly in Asia, but it spread to countries outside of Asia due to its multiple uses, such as a food source for humans, a soil fertilizer, animal feed, and even for medicinal purposes.[7,8] Mung bean is an important legume crop that can be cultivated twice a year, in the spring and autumn.Its seeds are more palatable, nutritious, easily digestible, and non-gassy.It contains 24.7% protein, 0.6% fat, 0.9% fiber, and 3.7% ash [9].The aim of the study is to isolate and diagnose Rhizobium bacteria associated with mung bean plants and testing the efficiency of the isolates in the promotional criterion of isolates for plant growth.

Sample Collection
35 samples of the root nodules of the Mung bean plant were collected on 14/6/2022 from various areas of Iraq : Salah al-Din Governorate.The plants were gently uprooted with their root system and up to a depth of 30 cm within Rhizosphere to preserve the nodules, if any.The samples were then placed in sterilized plastic bags (made of polyethylene) and transported to the laboratory for isolation and diagnosis.

Isolation of Rhizobium Bacteria
The roots were washed under calm tap water to remove the attached soil.Active pink nodules were selected, separated from the root system, and placed in sterilized Petri dishes.They were then washed several times with sterilized water, and surface treatment was carried out using 95% ethyl alcohol for 5-10 seconds, followed by washing with sterilized water 4-5 times.The nodules were then immersed in 0.1% HgCl2 solution for 3-4 minutes, followed by washing with sterilized water at least 6 times.The nodules were transferred to new Petri dishes, crushed with a sterilized glass rod in sterile conditions to obtain a bacterial suspension.Then, 1 ml of the bacterial suspension was taken using a sterilized pipette and inoculated on YEMA poured into sterilized plastic dishes.The plates were then incubated at 28°C for 3-7 days [10].

Purification of Rhizobium Bacteria
Ten bacterial colonies that grew on YEMA medium after the incubation period were selected and streaked on YEMA again to obtain pure colonies for performing visual, microscopic, and biochemical tests for these bacteria [11].

Diagnosis of Rhizobium Bacteria
The process of diagnosing bacterial isolates was carried out phenotypically depending on several culture characteristics such as: shape, texture, color, convexity and transparency only [12] and microscopically using Gram stain [11] as well as motility test using the hanging drop method [13].

Congo Red Stain Test
This test was conducted to differentiate between Rhizobium and Agrobacterium bacteria at different concentrations by absorbing the stain.Rhizobia bacteria do not absorb the stain and remain white in color, while Agrobacterium stains red as a result of absorbing the stain [14].

Bromothymol Blue Stain Test
This test was used to identify the bacterial genus of the slow or fast-growing groups Rhizobium or Bradyrhizobium by determining the ability of the bacteria to produce acid or base by changing the color of the medium containing the stain from green to yellow or blue.The medium was prepared with different concentrations, sterilized with autoclave, then inoculated with the isolated bacteria by streaking method, and incubated at a temperature of 28°C for 2-7 days, then the results were recorded [13].

Hofer Alkaline Test
This test is a way to distinguish between the genus Rhizobia and Agrobacterium because the genus Agrobacterium has the ability to grow at high levels of pH (pH 11), while Rhizobia bacteria cannot grow at these levels, as the medium was inoculated with a pure smear of the growing colonies and then incubated, the dishes were kept at a temperature of 28°C for 48 hours and after growth the results were recorded [15].
 Tests For Oxidase, Catalase,Indol, and Urease: These tests were performed according to Collee et al (1996) [11] and Aneja et al (2005) [16]. Test The Efficiency Of Isolates In Producing Chelating Compounds: The test was conducted according to the method described by Payne [17]. Test The Efficiency Of Isolates In Producing Indole Acetic Acid : The method described by Patten and Glick [18] . Test The Efficiency Of Isolates In Phosphate Solubilization: The ability and efficiency of isolates in phosphate solubilization are quantitatively measured using Pikovskaya liquid.This medium was used to assess the ability of bacteria to solubilize phosphates according to the method of Pikovskaya [19].The supernatant was then taken to estimate the soluble phosphorus using the spectrophotometer according to the method of Olsen and Watanbe [20].

Molecular Diagnosis
The isolates were molecularly identified by amplifying the 16S rRNA gene using the PCR technique with a pair of primers and the Universal Primer to identify the selected isolates for the ITS region ]11[.Table 1.General primers used in the study with their sequences.3 and 4.  The concentration and purity of the extracted DNA was estimated using a (Nano drop Spectrophotometer) by taking 1 microliter of DNA samples (DNA Stock solution) after centrifuging the mixture for five seconds to ensure that the liquid droplets on the surface of the tube wall, then put it in the designated place in the device and take a reading of the concentration and purity.

Electrophoresis on Agarose Gel
Mixing 3 µl of processor loading buffer (Intron/Korea) with 5 µl of DNA presumed to be ready for electrophoresis with (loading dye), and after mixing, loading was carried out into the gel orifices.Then an electric current of 7 V\C2 was exposed for 1-2 h until the dye reached the other side of the gel.The gel was tested by UV source with 336 nm after placing the gel in a bath containing 30 µl of DNA extract solution with red dye and 500 ml of distilled water.

Results and Discussion
Table 4 shows that most isolates have grown within 24-48 hours, indicating that they belong to the fast-growing Rhizobium species, except for one isolate that grew in 5-7 days, which belongs to the slow-growing Bradyrhizobium species.All ten isolates were spherical, smooth, mucoid, and had a creamy, white, or yellow color.These results are consistent with [24], [25] .theresults in Table 5 show that all isolates were gram-negative and motile after 24 hours of growth on Yeast Extract Manitol Broth medium (YEMB) using the hanging drop method.This finding is consistent [26] .Table 5 shows the variability of isolates based on their ability to absorb the Congo red dye, where eight isolates appeared as light pink and two as pink, This result is consistent with [1] in their study on Bradyrhizobium vignae bacteria isolated from the roots of the cowpea plant.Most isolates changed the color of the medium from green to yellow when grown on a medium containing bromothymol blue, indicating that they produce compounds that increase the acidity of the medium, and these belong to fast-growing groups (Rhizobium) except for one isolate (M6) that changed the color of the medium to blue, indicating that it produces compounds that increase the alkalinity of the medium, and these belong to slow-growing groups (Bradyrhizobium), This is consistent with [27] in their study on Rhizobium bacteria isolated from the root nodules of Faba Bean.All isolates gave a positive result for the catalase test, indicating their ability to break down hydrogen peroxide, leading to the release of oxygen bubbles, which is consistent with [28], in their study on Rhizobium bacteria isolated from the roots of nine legume plant species.Most isolates also gave a positive result for the oxidase test, which is consistent with , [29] in their study on Rhizobium leguminosarum bacteria in soybean plants.Most isolates gave a negative result for the indole test, which is consistent with, [30] in their Identification of B.japonicum bacteria isolated from soybean and cowpea plants.All isolates gave a positive result for the urease test except for one isolate, which is consistent with [31], in their study on Rhizobium leguminosarum bacteria isolated from the roots of the bean plant.Most isolates were negative for the alkaline Holfer test except for one isolate, which is consistent with [32], in their diagnosis to Rhizobium bacteria isolated from Vigna unguiculata plants.The table 6 shows that isolates (M3, M4, M5, M6,M7) have high ability to Iron chelate by their dense growth, while the rest of the isolates showed medium ability to Iron chelate except for isolate (M8) which showed weak ability to Iron chelate, Bacteria that do not grow on this medium are unable to produce chelating compounds and cannot pull the iron ion from the medium.However, bacteria that produce these compounds are capable of chelating and transporting the ion from the 2,2-dipyridyl compound inside the bacterial cell for use in metabolic activities ]33[.The differences in the isolates' ability to produce chelating compounds may be due to genetic variation and differences in growth and the amount of iron that they solubilize from the medium.These results are somewhat consistent with ]1[ in their study on Bradyrhizobium vignae bacteria, which showed that all bacterial isolates have the ability to grow on this medium, with four isolates having high ability to Iron chelate by their dense growth, while the rest of the isolates showed average ability to Iron chelate except for one isolate which showed weak ability to solubilize iron.Table 6 shows that all isolates were efficient in phosphate solubilization, but with varying ratios.Isolate (M6) belonging to the species Bradyrhizobium japonicum gave the highest phosphate solubilization with 39.504 mgP.L -1 , followed by isolate (M3) belonging to the species Rhizobium leguminosarum which gave a solubilization of 23.723 mgP.L -1 .The lowest solubilization was recorded for isolate (M2) which gave a solubilization of 13.863 mgP.L -1 .The reason for the ability of Bradyrhizobium bacteria to solubilize phosphate may be attributed to their ability to produce low molecular weight organic acids such as citric acid, gluconic acid, lactic acid, formic acid, and acetic acid, which act to chelate iron, calcium, and aluminum ions associated with phosphate due to their hydroxyl and carboxyl groups.This leads to the transformation of mineral phosphorus into forms ready for plants ]33[.These results are somewhat consistent with ]33[ , in their study of Bradyrhizobium manausense isolated from bean plants.The table 6 shows that the (M6) isolate surpassed the other isolates in its high ability to produce indole with a rate of 21.5 micrograms/mL -1 , followed by the( M3) isolate with a rate of 17.2 micrograms/mL - 1 , then the (M5) and( M7) isolates with rates of 13.2 and 13.6 micrograms/mL -1 respectively, and the least efficient isolate in producing indole was the (M10) isolate with a rate of 7.2 micrograms/mL -1 .The reason for the variation in the amount of indole produced by different types and strains may be attributed to genetic variability of these isolates since they belong to different types, which affects their biological properties, including their secretion in the growth medium.This result is somewhat consistent with ]31[, in their study on R. leguminosarum bacteria isolated from pea plants, where they found that the R15 isolate gave the highest efficiency in producing indole with a rate of 28.4 micrograms/mL, followed by the R19, R13, and R17 isolates which gave indole production rates of 26.1, 24.5, and 21.9 micrograms/mL -1 , respectively, and the R23 isolate gave the least ability to produce indole with a rate of 13.0 micrograms/mL -1 .Table 6.Efficiency of bacterial isolates in phosphate solubilization, indole production, and iron chelation.

Results of Molecular Diagnosis
Table 7 shows the Molecular diagnosis for five competent isolates was performed in the production of Indole acetic acid, production of chelating compounds, and phosphate solubilization, the results of molecular diagnosis showed that two isolates belong to Rhizobium bacteria.Fig. 1 shows the template of the DNA segment used in the amplification process and Fig. 3 shows the product of gel electrophoresis for extracting DNA from ibosomal region 35 to 630 showed 99.50% similarity with the bacterial species Rhizobium leguminosarum, isolate OTU21_I and ibosomal region 101 to 867 showed 99.48% similarity with the bacterial species Bradyrhizobium japonicum strain:NK5 and ibosomal region 31 to 795 showed 98.96% similarity with bacterial species Bradyrhizobium japonicum strain A3 that were recorded in the NCBI National Genetic Center with the number (OP975690.1)for Rhizobium leguminosarum and (OP975688.1)for Bradyrhizobium japonicum on 9/12/2022, which is the first strain recorded in gypsum soils in Iraq.Fig. 3 ,4 shows Registration form for the identified bacterial isolate Rhizobium leguminosarum and Bradyrhizobium japonicum .

Conclusion
From 35 samples of the root nodules associated with mung bean plants, ten samples isolated by growing them on yeast extract mannitol agar (YEMA), and the phenotypically pure isolates were diagnosed based on the culture, microscopic and biochemical characteristics and testing the promotional criterion of isolates.molecular diagnosis was performed for five competent isolates in the production of Indole acetic acid, production of chelating compounds, and phosphate solubilization.result shows that two isolate belong to Rhizobium bacteria, it was found that the (M6) isolate, which showed the highest ability to produce indole acetic acid, phosphate solubilization, and produce

Figure 1 .
Figure 1.Templates for the DNA fragment used in the amplification process.

Figure 4 .
Figure 4. Registration form of the identified bacterial isolate Bradyrhizobium japonicum.

Table 4 .
Some morphological and microscopic characteristics of bacterial colonies isolated from the root nodules associated with the mung bean plant.

Table 5 .
Some biochemical tests for bacterial isolates obtained from the root nodules of Mung bean plants.

Table 7 .
Molecular diagnosis of bacterial isolates based on the percentage of matching 16S rRNA gene sequences with bacterial strains registered in the global genetic bank at NCBI.