Isolation and Characterization Identification of Edophytic Nitrogen-Fixing Bacteria from Peanut Nodules

This work was carried out to isolate and perform molecular identification and selection of endophytic nitrogen-fixing bacteria (ENFB) to be utilized as biofertilizer. In this research, nodulous samples of peanuts were collected from inside dyke areas, namely, Phuoc Hung of An Phu, An Giang, Vietnam. Ten colonies were isolated from nutrient agar plates containing YMA's medium. All isolates were rod shaped, Gram negative, and no spore creation. Biochemical tests indicated that they were obligate aerobes, catalase, oxidase, urea hydrolysis, well motile ability, and no nitrate reduction. The salt tolerance observed that most survived at 0.5% and 2% salinity (except Enterobacter cloacae subsp. dissolvens strain LMG 2683), while at 4%, only 3 isolates (Bacillus aryabhattai strain CM44, Enterobacter asburiae strain IIWM-JS-07L, and Bacillus songklensis strain KCa6) and at 5% only, 2 isolates survived, namely, Enterobacter asburiae strain IIWM-JS-07L and Bacillus songklensis strain KCa6. The result showed that most of ten ENFB strains could adapt to the range of 25°C and 45°C (except Enterobacter cloacae subsp. dissolvens strain LMG 2683 and Enterobacter mori strain cjy13 at 25°C). Out of ten isolates, three were finally selected for the next studies, which potentially have N-fixing ability and are utilized as biofertilizer in agricultural cultivation.


Introduction
Te agricultural soil plays an important role in global food production; as a result, crop soils are intensively farmed and planted to maximize yields.Farmers use high inorganic fertilizers in large quantities for many continuous years, which are really hazardous to the health of the crop soil and the quality of agricultural products [1].ENFB species in peanut root nodules have been studied in order to understand the symbiotic relationship for nitrogen fxation and mutual benefts [2,3].Endophytic nitrogen-fxing bacteria play a crucial role in crop growth by promoting plant biomass and yield, helping to meet the growing global demand for food as the population increases [4,5].Te root nodules of legumes are formed through a symbiotic relationship with ENFB called as rhizobia.Tese nodules have specialized structures and functions that facilitate the conversion of atmospheric N 2 into plant-usable forms such as NH +  4 and NO − 3 by the ENFB strains through a process called as biological nitrogen fxation.Te ENFB utilization for raising the crop yield is an economic and sustainable method, which reduce the utilization of inorganic fertilizers and pesticides [6].Endophytic nitrogen-fxing bacteria are popularly used as natural nitrogen fertilizers, promoting sustainable agricultural practices [7].Many prior research studies have proven that the peanut plant associated with ENFB strains could improve farmland fertility by fxing air nitrogen [8,9].Peanuts are able to take the nitrogen of air by the symbiotic relationship with ENFB, which resides inside peanut roots' nodules [10].Symbiotic N-fxation between rhizobia and legumes plays a crucial role in the ecological interaction between soil and crops, and it determines nutrient composition, biodiversity, and the succession of subsequent generations.Tey annually contribute around 100-290 million N tons to farmlands and crops.Tis natural N resource supports natural ecosystems, enhances plant growth, and holds global agronomic importance.In addition, the bacteria have increased the diversity of legumes due to this nitrogen fxation ability [8,10].Particular signifcance for the interaction between plants and biology is the legumerhizobia reciprocity, which fx N 2 sources of air to reclaim carbon supplied by legumes.Tis beneft interaction ensures regularly accurate nutrient cycles in natural ecosystems by supporting nitrogen in an agricultural cultivation [11].Te increasing cost of using various types of chemical fertilizers and the negative impacts of pesticides have led to reduced profts and quality of agricultural products.Tis problem requires a solution through the use of organic products and natural plant protection methods.Te use of plant growthpromoting bacteria to make plants healthier and improve soil quality has become an attractive method for developing sustainable farming systems due to their unique properties for the environment, production, low cost, and reduced consumption of nonrenewable fertilizer energy [12,13].From the abovementioned reasons, this study frst isolated and characterized native ENFB from peanut nodules.Tese selected strains could become a base product to improve crop productivity and raise soil fertility.

Nodule Collection.
Nodule samples of peanuts were from peanuts plants in Phuoc Hung commune, An Phu district, An Giang Province.Nodules, which were taken from peanut roots, were cut from the tumor using scissors and then the surface of the nodules were disinfected with CaOCl 2 solution (4%) for 180 seconds before being cleaned with fresh water for 10 seconds.Te collected nodule samples were in the size of 02 -03 mm in diameter.Te light or pink nodules indicated that a community of nitrogenfxing bacteria were acting and living inside [14,15].Later, nodules were soaked in 70% Alc.solution for 3 minutes and again washed with sterile distilled water.Te clean nodules placed into an Eppendorf, containing sterile distilled water to form suspension. Te above suspension was diluted from 10 − 2 to 10 − 6 and inoculated into 3 Petri dishes of YMA medium.Te plates after inoculation were incubated at room temperature for 4-5 days, followed by observation for the growth of colonies, recording for the results and inoculation and purifcation [16].

ENFB Isolation.
Te nutrient medium composition of YMA consisted of yeast extract, mannitol, K 2 HPO 4 , MgSO 4 , NaCl, agar, pH � 7, and adding Congo red solution to obtain the 25 mg•L − 1 content.YMA medium plates containing ENFB colonies were incubated at 28 °C for 4 days.It was continued by streaking the separated isolates until forming completely pure colonies from white to pink and malleable and shimmery.Te colonies of ENFB were then extracted from most other soil bacteria by fltration through YMA [14,15].
Te isolated ENFB genus was further classifed through a number of selective media since most of the ENFB strains were Gram-negative and rod-shaped bacteria.
(i) YMA-BTB medium: this can identify ENFB colonies fast because of turning agar to slowly blue color (ii) GPA-BCP medium: in this medium, ENFB strains poorly grew on because of high pH, used for checking the isolate purity [17].(iii) Lactose agar medium: this medium was to check the yellow ring around the colonies after being fooded with reagent of Benedict, confrming the ENFB presence because of converting lactose to 3ketolactose.It was called as the keta-lactose test [18].(iv) Hofer's alkaline medium: this medium was used for ENFB strains isolation, which could grow at high pH such pH > 6.5 [19].(v) N-free medium: this medium was used by isolating free-living ENFB strains, which could utilize air N to form protein synthesis.In this research, it was used for testing colonies, which could develop N-free medium or not [20].Checking Biochemistry was conducted using the ASM technology [21].
As shown in Figure 1, three pure colonies for endophytic bacterial genera were isolated from peanut nodules, which were selected as potential endophytic bacterial isolates with distinct morphological and colony color.Te isolation and genomic identifcation of endophytic bacterial genera from peanut nodules by the YMA nutrient medium was conducted for the identifcation of isolates and Gram symbols for identifcation of their biological properties.

16S rDNA Sequencing.
Pure ENFB colonies were placed in Eppendorf tubes for DNA extraction, which were used by the GeneJET Genomic DNA Purifcation Kits from Termal Scientifc ™ .Te 16S rDNA genes of the isolates was am- plifed by PCR using the primer set 20F 5′-CTACGGCAA GGCGACGCTGACG-3′ and 1500R 5′-GGTTACCTTGTT ACGACTT-3′ [16,22].Te sequence data were analyzed using MEGA software and compared to the most similar sequence in GenBank using the BLAST technique with an identity threshold of greater than 99.70% [23].

ENFB Characterization.
Te ENFB species were purifed and characterized by Gram staining, catalase and oxidase tests, and macroscopic and microscopic observations.Teir growth on YMA containing bromothymol blue was observed as well.In addition, the isolated species were tested for their resistance to heat and salinity, which are main factors for screening ENFB for the introducing an aim to them into degraded soils.

Termal Tolerance.
To determine the optimal incubation temperature, a set of 12 tubes containing YMA medium were prepared.Four tubes were prepared for each temperature and incubated at 25, 37, and 45 ± 1 °C in an incubator.Te growth of bacterial colonies was daily monitored for 7 days.Te thermos tolerance test of the isolated ENFB species was performed, which were streaked on YMA agar plates and incubated at temperature ranging 2 International Journal of Microbiology from 25, 37 and 45 °C.Te experiments were performed in quadruplicate for each species and the growth of colonies was recorded for one week [24].

Isolation and Characterization Identifcation.
Nodulous samples were collected from Phuoc Hung commune, An Phu district, An Giang, Vietnam.Tese collected nodules were isolated by the new technologies for ENFB [25].Te isolation and genomic identifcation of rhizosphere N-fxing bacteria from peanut nodules by the YMA nutrient medium was conducted for the identifcation of isolates and Gram symbols for identifcation of their biological properties.Table 1 presents the identifed species, noting that all isolates were negative for Gram staining after fooding.Te sequencing results (Table 2) showed that the similarity of the ten selected species was ≥99.5% compared to the standard ENFB species.Te NCBI database (https://www.ncbi.nlm.nih.gov/genbank/) was used to identify their highest similarity.
Table 3 illustrates the adaptability of ten ENFB strains to the temperature range of 25-45 °C, within which they were capable of surviving and growing.Notably, the optimal temperature range for their development fell between 37 and 45 °C.Excessive or insufcient temperatures in agricultural environments can adversely afect plant growth and nodule formation, ultimately compromising the nitrogen-fxing efciency of ENFB [26].YMA medium containing diferent NaCl concentrations (0.5%, 2%, 3%, 4%, and 5%) were inoculated at 28 °C for 48 hours.Te isolates grew in the diferent NaCl concentrations, which were then observed and compared together (from low to high NaCl concentrations).Te salt tolerance test observed that most survived at 0.5% and 2% salinity (except Enterobacter cloacae subsp.dissolvens strain LMG 2683 and Enterobacter mori strain cjy13), while at 4%, only 3 isolates (species: B2B, V1 and 18) and at 5%, only 2 isolates survived, namely, V1 and 18.Most of ten ENFB strains could adapt to the range of 25 °C and 45 °C (except Enterobacter cloacae subsp.dissolvens strain LMG 2683 and Enterobacter mori strain cjy13 at 25 °C) [27].In this study, the pH efect of selected colonies on the YEMA semisolid medium (acidifed with H 2 SO 4 ) was investigated after 5 days of incubating.Te results showed that seven out of ten selected strains exhibited weak growth at pH 4.5 (except for B2B, V1, and 18).Ten selected species grew well at pH � 7.However, the colonies of B2B, V1, V2, and 18 were able to survive on pH 8.5 (Table 3).
Figure 2 shows that the three selected species, including Bacillus aryabhattai strain CM44, Enterobacter asburiae strain IIWM-JS-07L, and Bacillus songklensis strain KCa6, exhibited remarkable characteristics when being tested for their ability to withstand heat, salinity, and other conditions.Furthermore, they shared a high degree of similarity with high nitrogen-fxing species.Tese species were selected based on previous screenings and were represented in the phylogenetic tree.Te phylogenetic determination used three selected isolates with reference neighboring sequences from the previous database.A close relationship of these branches was closely grouped together with the genera of Bacillus aryabhattai strain CM44, Enterobacter asburiae strain IIWM-JS-07L, and Bacillus songklensis strain KCa6.

Discussion
On Hofer medium, which had a high pH, the ability of selected species to grow well was considered as a key factor in assessing its potential to belong to the group of ENFB species [28].All ten obtained strains with clear pink colonies on YMA.It is a main characteristic of Rhizobium strain, already explained by Somasegaran and Hoben [15].Te test of GPA-BCP medium aimed to check a culture purity of a highly nutritious medium.Te results, as shown in Table 1, showed that all ENFB isolates were signifcantly developed on this medium.On contrary, other genus were weakly developed [29].All identifed ENFB species were checked for the nitrogen-fxing ability by Burk medium (without N) to determine their genera size.Tis result represented that isolated species developed well on agar medium (without nitrogen).Tese ENFB species were the highest priority selection to study on the feld experiments [30][31][32].Te tests of oxidase and catalase showed that all rhizobial strains had positive results.Tese results were consistent with previous studies revealing that rhizobial strains usually give positive results for the oxidase and catalase reaction [33,34].Te test of chemical traits showed diferent results dependent on each rhizobial species (Table 4).However, theses result proved that diferent factors of ENFB species were highly afected by their living environment [35,36].Te 16S rRNA sequences of the ten selected strains, as presented in Table 2, were derived from the rhizosphere of peanut plants.Tis analysis, along with other cutting-edge molecular techniques, has facilitated the discovery of numerous new ENFB species.Tese fndings have signifcantly expanded our knowledge of ENFB diversity and their valuable attributes.Overall, learning these new techniques is a means to enhance the reliability and efectiveness of molecular research and applications.[37].Te selected ENFB species could have the high adaption of NaCl concentration up to 3-4% [24] or even to 5% (w/v) NaCl [38].However, NaCl concentration was below 1% (w/v), which was suitable for most ENFB species.Ten obtained species were suitable in pH from 5 to 8 [37,39].International Journal of Microbiology

Conclusions
Ten ENFB species were isolated, identifed, and tested by various biochemical and molecular assays.Tree selected potential species consisted of Bacillus aryabhattai strain CM44, Enterobacter asburiae strain IIWM-JS-07L, and Bacillus songklensis strain KCa6, which were tested for the abilities of thermal, saline pH tolerance, and for their categorization into groups to be used for further feld studies to the ultimate goal of developing inoculants in future.Understanding ENFB and their host plant origins has enabled us to make more extensive and confdent use of these positive species, as long as their optimal conditions are still well concerned.

Figure 2 :
Figure2: Phylogenetic tree of partial 16S rRNA sequences of ENFB strains isolated from peanut nodules along with the sequences from selected references strains.Te tree was constructed by the neighbor-joining method using MEGA 11.Te scale bar corresponded to 0.050 substitutions per nucleotide position.Numbers on the branches were bootstrap percentages.GenBank accession numbers are shown above.