Wulandari Et Al 2024
Wulandari Et Al 2024
Wulandari Et Al 2024
https://doi.org/
Received: 25 October 2023, Revised: 31 January 2024, Accepted: 7 February 2024, Published: xx May 2024
Abstract
Rhizobia are bacteria that symbiosis with host plant, as shown in the root nodules formation, and
provide nitrogen that can be absorbed by plants in greater quantities than rhizobacteria. Available Nitrogen,
which absorbed by plants, is the essential requirement for plant growth because its role in increasing yield
and quality, hence it is needed in greater quantities than other nutrients. The study aimed to determine the
macroscopic and microscopic diversity of rhizobia isolates from the groundnut nodules and their potential
as PGPRs, and to identify 16S rRNA isolates with the best potential as PGPRs molecularly. The methods
used were isolation from root nodules, screening of PGPR potential, molecular identification based on the
16S rRNA gene, and phylogenetic analysis to determine their kinship. Based on the isolation results, 17
Gram-negative isolates were obtained white to pink or orange color on AG media with various colony
characteristics in terms of shape, margin, elevation, and texture. KT 20, which was selected as rhizobia
isolate with the best potential as PGPR, has ammonium concentration of 23.12 ppm, synthesizes IAA with
a concentration of 10.36 ppm, and phosphates solubilization activity, although its ability to synthesize
proteases is low. The results of molecular identification of 16S rRNA showed that KT 20 belongs to the
Rhizobium genus with a similarity of 99.48 % and bootstrap value of 96 %.
Keywords: Arachis hypogaea, IAA synthesize, Nitrogen fixation, Proteases, Phosphate solubilization,
Rhizobium sp.
Introduction
Plant growth promoting rhizobacteria (PGPR) are rhizobacteria (soil bacteria on the root surface and
surrounding) that support plant growth through their various activities, including nitrogen fixation,
phosphate solubilization [1], synthesizing phytohormones such as indole-3-acetic acid (IAA) [2], as well
as synthesizing lyses enzymes, such as proteases [3]. There is a huge number of bacterial species that
identified as PGPR, at least 15 genera, including Bradyrhizobia, Sinorhizobia, Rhizobia, Devosia,
Azorhizobia and Ochrobactrum [4]. However, their ability as PGPR were limited to the specific host plant
[5] and some species find it difficult to adapt when studied in vivo.
Rhizobacteria that have symbiosis with legumes are called rhizobia. The symbiosis between rhizobia
and host was shown by root nodules formation on legume plants [6]. Symbiotic bacteria were able to
provide nitrogen that can be absorbed by plants in greater quantities than rhizobacteria. Furthermore,
Meghvansi et al. [7] stated that indigenous rhizobia capable to associate with other soil microbes and
adaptable to extreme conditions with ease.
One of the plants that is adaptable too is groundnuts (Arachis hypogaea L.), one of the legumes that
is produced commercially [8]. This plant has been cultivated at least in 118 countries, including Indonesia
Trends Sci. 2024; 21(xx): xxxxx 2 of 14
[9]. In 2020, FAOSTAT reported that the groundnut crop harvest area of 345,000 tons from a planting area
of 364,000 ha and a total production of groundnuts with shell of 860,000 tons in Indonesia.
Moreover, rhizobia which infect groundnut has a different way of nodulation than rhizobia in general.
These rhizobia do not develop infection threads, but enter through lateral root crack on the epidermis, then
immediately go to cortical cells by a process that like an endocytosis [10]. Due to the presence of “crack
entry” method, these rhizobia symbiotic with non-legume plants more easily [11].
Based on these, the research related to bioprospecting was conducted. Bioprospecting is the process
of exploring materials from various organisms to obtain valuable products as the need for the material
increases, including in agroindustry [12]. Bioprospection plays a significant role in the conservation and
sustainable use of biodiversity. According to that, this study aimed to determine the macroscopic and
microscopic diversity of rhizobia isolates from the groundnut nodules and their potential as PGPRs, and to
identify 16S rRNA isolates with the best potential as PGPRs molecularly.
The PSI values were classified (Table 1) according to the categories used by Halimursyadah et al.
[23].
Proteases activity
Analysis of protease activity conducted by refers to the study of Mehta et al. [26]. After growing
isolates in AG broth media for 3 days, 5 𝜇L of each isolate culture was drip on paper disc and it was placed
on 12.5 g/L skim milk agar (SMA) media for 24 - 48 h [27]. Then, the clear zone that appears was observed
and calculated to determine the proteolytic index (PI) using the equation by Lim et al. [28], as follows:
The PI values were classified according to the categories used by Dewiyanti et al. [29] (Table 2).
PI value Efficiency
<1 Low
1-2 Moderate
>2 High
Components Concentration
GoTaq PCR Mix 2X
Primer 27F 100 pmol/𝜇L
Primer 1492R 100 pmol/𝜇L
DNA Templates 20 ng
ddH2O
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Phylogenetic analysis
The isolate sequences were beread and contig with BioEdit version 7.0.5.3, then being compared with
gene sequence data on the National Center for Biotechnology Information (NCBI) website
(https://www.ncbi.nlm.nih.gov/) using the basic local alignment search tool-nucleotide program (BLAST-
N). A phylogenetic tree was created with MEGA XI software under the Neighbor-Joining Tree construction
using the bootstrap 1,000 method [31].
Microscopic characterization
Twenty-seven isolates that had been selected macroscopically were re-selected through Gram
staining, thus proving that all isolates were Gram negative, except for KT 6. This was in accordance with
the stated by Etesami [39] that, N-fixing bacteria in nodules are Gram-negative bacteria. Based on the Gram
stain’s results, 26 isolates were Gram negative which was characterized by a pink color with 69.23 % being
rod-shaped. Thairu et al. [18] stated that, Gram-positive and Gram-negative bacteria are differentiated
based on the thickness of their cell wall (peptidoglycan) and permeability of their membranes. Gram-
positive have thick peptidoglycan as their cell wall, thus they are kept the crystal violet-iodine complex
when decolorized. While bacteria Gram negative has a thin peptidoglycan and lipids-rich
(lipopolysaccharides) that cause the cell wall is damaged and loses the crystal violet-iodine complex, then
gain safranin.
such as NH4+ and NO3−. Out of the 26 isolates, 17 showed positive results for fixing nitrogen, namely KT
1, KT 2, KT 3, KT 7, KT 11, KT 13, KT 14, KT 15, KT 16, KT 17, KT 18, KT 19, KT 20, KT 21, KT 22,
KT 24, and KT 25, which indicate by the appearance of blue color after incubation for 7 days (Figure 1).
In NFB media, there is bromothymol blue reagent whose color is affected by pH. Thus affect the
appearance of blue color, since the isolates fix nitrogen and then formed ammonium, which is alkaline. This
statement supported by Shimada and Hasegawa [42] that, the green color could change to blue according
to the level of acidity in the NFB medium, so that it is used as an indicator of nitrogen fixing bacteria.
(a) (b)
Figure 2 Phosphate solubilization test (a) control and (b) positive (KT 20).
Based on the analysis results, KT 2, KT 3, KT 7, KT 11, KT 13, KT 14, KT 17, KT 18, KT 20, KT
21, and KT 24 have a good ability with PSI value is more than 2.16. Kumar et al. [44] stated that, one of
the PGPR’s roles is to provide the phosphate needed on plant growth by assimilating insoluble phosphate
to soluble and absorbed by plants, because most of the P is absorbed in anion forms, i.e. HPO4 2− and
H2PO4−.
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This result is supported by Gang et al. [45], the pink color appears as a positive reaction from the
formation of indole compounds as a result of tryptophan metabolism, the synthesized IAA forms a complex
with Fe3+ from Salkowski reduction. According to Chandra et al. [46], the ability to synthesize IAA is also
necessary for plants, because IAA increases the root length by enhance the number of branches, lateral
roots, and root hairs, thus helping the absorption of nutrients from the surroundings.
Protease activity
Furthermore, 12 out of 17 rhizobia isolates showed the ability to synthesize proteases. This is
indicated by the formation of a clear zone around the colony on SMA media because the bacteria produce
enzymes that breakdown proteins in SMA media and forms a clear zone (Figure 4). According to
Ramadhan et al. [47], the clear zone that formed around the colony indicated that the bacteria growing on
SMA media had broken down proteins in skim milk into amino acids and peptides by producing protease
enzymes.
Based on the analysis results, at most KT 22 has a good ability with PI value is more than 1.00. Bhat
et al. [48] stated, the protease synthesized by PGPR is also known as plant-growth promoter, as they
degraded pathogenic cell walls, which composed of proteins, thereby inhibiting these plant pathogens.
According to the concentrations of NH4+ and IAA, KT 20 has much better potential in fixing nitrogen
(Figure 5). This statement is supported by Shimada and Hasegawa [42] that the color change between
yellow, green to blue in media with bromothymol blue follows changes in the pH of the media, so that it is
along with the concentration of the ammonium produced. Shoukry et al. [49] also reported that the
concentration of IAA is along with the level of red color as a reaction between IAA and the Salkowski
reagent. According to Simon et al. [50], rhizobia are soil microorganisms that can infect the roots of the
Leguminosae family and fix nitrogen, thus it can be used by plants.
30
Concentrations (ppm)
25 23.12
20 17.72
15 10.36 8.96
10 6.53
4.55
5 1.8 2.81
0
-5 KT 12 KT 19 KT 20 KT 24
Isolate
Ammonium IAA
KT 20 was chosen as the superior rhizobia isolate for molecular identification of 16S rRNA, although
KT 24 had a better ability to synthesize IAA and increase soluble phosphate, because the legume plant
needs of N-available was very high. Kabir et al. [51] and Xu et al. [52] stated that, N-available is the most
important condition for plant growth and needed in greater quantities than other nutrients. Even though the
Leguminosae family, such as the groundnut is able to fix as much as 40-80 kg/ha N in a year, these plants
still require about 86 - 92 % N (125 - 178 kg/ha N) fixed by microbes.
DNA
KT 20
Marker
1,500 bp 1,500 bp
250 bp
The bootstrap value between KT 20 and Rhizobium sp. strain KNR2.6 was 96 % (Figure 7) indicates
the accuracy level of the phylogeny. This statement is supported by Hillis and Bull [60], a bootstrap value
of more than 95 % with a limit above 70 % showed a credibility.
Since the BLAST-N of KT 20 showed that the query cover, percent identity and E-value has the same
score for 9 species belonging to the Rhizobium genus, then KT 20 is also identified as a species belonging
to the Rhizobium genus. This isolate has a circular colony-shape, undulate edge, raised elevation, and shiny
light pink color. This result is supported by Silva et al. [61], Rhizobium sp. is well known as a rod-shaped
Gram-negative bacterium that has a circular in shape with undulate or entire edge and raised or flat elevation
(Figure 8). In addition, the results of a study by Dinkwar et al. [62] showed that the color of some
Rhizobium sp. are shiny whitish pink. In addition, this bacterium also has the ability in phosphate
solubilization, IAA synthesize, and protease synthesize as some of the characteristics required by PGPR.
These results are supported by Sridevi and Mallaiah [63] who reported that the phosphate solubilization
ability of Rhizobium sp. strain 26 was the smallest compared to 40 rhizobia isolated from root and stem
nodules of Cassia absus, Vigna trilobata, and 3 strains of Sesbania sesban. Kumar and Ram [64] also
obtained Rhizobium sp. able to solubilize phosphate, namely Rhizobium sp. strains 103-JX576499 and
Rhizobium sp. strain MRR106-KC428655. While, the ability of IAA synthesize is supported by Shokri and
Emtiazi [65] and Ghosh and Basu [66] who obtained Rhizobium sp. from the root nodules of Phaseolus
mungo var. PU30 synthesize IAA. In addition, although not many studies have reported the ability of
Rhizobium sp. in synthesizing proteases, Purwaningsih et al. [67] reported that 8 out of 10 isolates of
Rhizobium sp. isolated from Arachis hypogaea able to synthesize proteases.
Conclusions
There are 17 rhizobia isolates from the root nodules of groundnut with various macroscopic
characteristics. The potential PGPR that selected is KT 20, which has a circular colony-shape, undulate
edge, raised elevation with a glistening light pink. KT 20 was chosen because it has the best nitrogen
fixation ability and able to solubilize phosphate, synthesize IAA, and synthesize protease. This isolate
belongs to the Rhizobium genus with a similarity of 99.48 % and bootstrap value of 96 %. For further
studies, it is necessary to conduct in vivo research and identify rhizobia-specific genes molecularly to ensure
that the isolates are rhizobia species which nodulation groundnut root nodules.
Acknowledgment
AB would like to thank Diponegoro University, Indonesia for the RPI Grant No. 753-
24/UN7.D2/PP/IX/2022 and WCU UNDIP Grant for Staff Exchange 2024. DW would like to thank
Diponegoro University for the Postdoc Grant No. 216/UN7.A/HK/XI/2023. In memoriam Dr. Rejeki Siti
Ferniah
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Highlights
• Isolate KT 20 (identified rhizobia) has excellent ability in fixing nitrogen, which plants needs the
most and legumes requires in large number
• Beside the ability of Isolate KT 20 to fix nitrogen, it also able to synthesize IAA, solubilize phosphate,
and synthesize protease
• The ability of isolate KT 20 as PGPR was equal or better than other rhizobacteria that has been
analyzed in other in vitro studies
Trends Sci. 2024; 21(xx): xxxxx 14 of 14
Graphical abstract