agriculture

Article
Enhancing Upland Rice Growth and Yield with Indigenous
Plant Growth-Promoting Rhizobacteria (PGPR) Isolate at
N-Fertilizers Dosage
Rahma Tia Harahap, Isnaniar Rahmatul Azizah , Mieke Rochimi Setiawati, Diyan Herdiyantoro
and Tualar Simarmata *

Department of Soil Science, Universitas Padjadjaran, Jatinangor 45363, West Java, Indonesia;
rahma21022@mail.unpad.ac.id (R.T.H.); isnaniara@gmail.com (I.R.A.); m.setiawati@unpad.ac.id (M.R.S.);
d.herdiyantoro@unpad.ac.id (D.H.)
* Correspondence: tualar.simarmata@unpad.ac.id

Abstract: Upland rice farming plays a crucial role in ensuring food security in Indonesia. This study
aimed to evaluate the impact of plant growth-promoting rhizobacteria (PGPR) isolates on the growth
of upland rice. The bioassay and pot experiments were conducted to select the capable isolates of
PGPR and to investigate the effect of the PGPR inoculant on the N fertilizer efficiency and agronomic
traits of upland rice. The bacterial isolates were identified through a biochemical analysis and tested
under controlled greenhouse conditions. The selected PGPR inoculant was formulated as a liquid
biofertilizer (LB). The three capable isolates were obtained to fix nitrogen, produce indole-3-acetic
acid (IAA), organic acid, and nitrogenase activity and were identified through a biomolecular analysis
as Delftia tsuruhatensis strain D9, Delftia sp. strain MS2As2, and Bacillus sp. The application of the
LB into the soil at a dose of 10 L ha−1 and 50 kg ha−1 N resulted in a grain yield of 29.81 g pot−1
and a relative agronomic effectiveness (RAE) value of 235.08%, signifying a significant improvement
over the conventional method. Several variables, including the number of grains, number of panicles,
Citation: Harahap, R.T.; Azizah, I.R.; root length, 1000-grain weight, population of nitrogen-fixing bacteria, and nitrogen uptake exhibited
Setiawati, M.R.; Herdiyantoro, D.; a strong correlation with the grain yield, accounting for 97.80% of the observed variation. These
Simarmata, T. Enhancing Upland
findings show the enormous potential of PGPR isolates, specifically of Delftia tsuruhatensis strain
Rice Growth and Yield with
D9, Delftia sp. strain MS2As2, and Bacillus sp., in significantly enhancing the upland rice output in
Indigenous Plant Growth-Promoting
Indonesia. Furthermore, the use of an LB as a biofertilizer in conjunction with nitrogen fertilization
Rhizobacteria (PGPR) Isolate at
provides a viable and sustainable way to increase yields and enhance the overall sustainability of the
N-Fertilizers Dosage. Agriculture
2023, 13, 1987. https://doi.org/
region’s upland rice farming systems.
10.3390/agriculture13101987
Keywords: liquid biofertilizer; N-fixer bacteria; agronomics traits; upland rice; drylands
Academic Editors: Salvadora
Navarro-Torre and Ignacio
David Rodríguez-Llorente

Received: 19 August 2023 1. Introduction

Revised: 1 October 2023 Rice (Oryza sativa L.) is the primary food consumed by more than half of the world’s
Accepted: 4 October 2023 population, and particularly in Indonesia. Around 98% of the Indonesian population
Published: 13 October 2023
consumes rice as a staple food [1–3]. Upland rice is one of the potential food crops that can
be developed in drylands, offering significant opportunities for food production. However,
upland rice production is still relatively small [4]. The increase in production can be
Copyright: © 2023 by the authors.
achieved through the application of biofertilizers.
Licensee MDPI, Basel, Switzerland. Biofertilizers or microbial inoculants are the most attractive of these alternatives
This article is an open access article because of their positive impact on plant growth as well as the environment [5]. In recent
distributed under the terms and years, the use of biological fertilizers to reduce the use of chemical fertilizers has become a
conditions of the Creative Commons public concern and is increasingly being applied. In general, biofertilizers do not provide
Attribution (CC BY) license (https:// nutrients to plants, but the microbes present in biological fertilizers can break down organic
creativecommons.org/licenses/by/ compounds in the soil into simple ionic forms that plants can absorb. These microbes also
4.0/). assist in converting the nutrients from unusable forms into forms that plants can effectively

Agriculture 2023, 13, 1987. https://doi.org/10.3390/agriculture13101987 https://www.mdpi.com/journal/agriculture

Agriculture 2023, 13, 1987 2 of 21

utilize for growth [6]. The association between the plants and symbiotic microorganisms
is involved in key functions at the ecosystem and plant levels, and the application of
microbial plant biostimulants (MPBs) is a sustainable strategy to augment plant growth
and productivity, even under abiotic stress conditions [7,8].
Plant growth-promoting rhizobacteria (PGPR) are a group of beneficial microorgan-
isms that are often inoculated as biological fertilizers. This group of bacteria presents
relevant properties to fix the essential plant nutrients such as nitrogen (N), thus are known
as N-fixing PGPR [9]. In addition to their ability in nutrient provision, PGPR also possess
several functional traits, such as the following: inhibiting soil pathogens by producing
HCN (hydrogen cyanide), siderophores, and antibiotics; improving plant tolerance to
drought, salinity, metal toxicity, and P dissolution; and producing phytohormones [10,11].
The ability of nitrogen-fixing bacteria to produce phytohormones is considered essential
because they influence root growth resulting in increased water and nutrient uptake. PGPR
also help in mitigating abiotic pressures (salinity and drought), reducing the impact of
heavy metals in the soil, and the biocontrol of plant pathogens [9,10]. Previous research
on PGPR biofertilizers demonstrated no significant difference between the 100% NPP
(nitrogen, phosphorus, and potassium) fertilizer dose and the 50% NPP dose when applied
to upland rice cultivation [12,13]. Moreover, numerous studies have consistently shown
that the inoculation of selected PGPR isolates has a significant positive impact on various
rice growth parameters, including the sprout percentage, shoot growth, and chlorophyll
content, when compared to the uninoculated control [14]. However, to apply the PGPR
isolates as biofertilizers effectively, compatible carrier materials are essential to facilitate
their successful transfer from the laboratory to the field [15–17]. Glycerol and molasses
are liquid carriers that are commonly used as the carbon sources for microbes during
incubation. These carriers play a crucial role in enhancing the survivability and efficiency
of rhizobacterial inoculants in biofertilizer formulations [18].
The purpose of the research was to evaluate the impact of different combinations of
liquid biofertilizers and nitrogen fertilizer doses on improving the growth characteristics
and yield components of upland rice.

2. Materials and Methods

The isolation of PGPR was performed using Okon’s selective media, which led to the
identification of a total of nine isolates. Nine selected isolates were obtained from five dif-
ferent ecosystems on Lombok’s Island, West Nusa Tenggara, Indonesia. These ecosystems
included the rainfed areas in Pujut, Central Lombok; the maize fields in Pringgabaya, East
Lombok; the mixed crop in Sambelia, East Lombok; the forest within the Mount Rinjani
National Park, Tetebatu, East Lombok; and the savanna regions in Sembalun, East Lombok,
and the characteristics of each were described in our published article [19]. Three capable
isolates were selected based on their growth-promoting properties, results of biochemical
tests, and their ability to enhance rice growth characteristics. The three capable isolates
were further identified using molecular methods, specifically 16S rRNA sequencing, to
determine their bacterial species. The identification process was conducted by Genetica-
Science using Bioedit software version 7.0.5.3 [20] and Molecular Evolutionary Genetics
Analysis (MEGA) version X [21].
A consortium of three capable isolates was inoculated into a liquid carrier, preparing
it for subsequent application in Situ Bagendit upland rice cultivation through a pot experi-
ment. The primary goal of this process is to optimize the growth characteristics and yield of
upland rice while simultaneously increasing the efficiency of nitrogen fertilizer utilization.

2.1. Bioassay and Biochemical Test

2.1.1. Bioassay Test
Bioassays were conducted on nine isolates obtained from drylands. The bioassay for
N-Fixing PGPR was performed by culturing the isolates on N-free Fahraeus’ medium [13].
Upland rice seeds were sterilized with a 0.2% HgCl2 solution for approximately 30 s,
Agriculture 2023, 13, 1987 3 of 21

followed by rinsing with 70% alcohol for about 1–2 min and washing with distilled water
three times before germination. Subsequently, the seeds were planted in 100 mL test tubes
filled with a liquid suspension of PGPR inoculum and Fahraeus’ medium in a ratio of 1:9
by volume. Each treatment was placed in the greenhouse of the Biotechnology Laboratory
at Padjadjaran University, where plant height and root length were observed on a weekly
basis. Shoot dry weight and root dry weight were analyzed by harvesting the entire rice
plant 28 days after planting. At 28 days after sowing, the above ground parts of seedlings
were still exhibiting some growth, and both the seedling dry weight and dry weight per
unit of seedling height had increased slightly [22]. Samples were carefully placed in small
envelopes and subjected to oven drying at a temperature of between 70 ◦ C and 80 ◦ C for
48 h, or until a constant weight was achieved. The dry weight of the shoots and roots was
then calculated. The bioassay was conducted over a period of 28 days to evaluate the ability
of rhizobacteria to enhance plant growth. The experiment followed a randomized complete
block design (RCBD) with 10 treatments and 3 replications, resulting in 30 experimental
units. The treatments consisted of the following different isolates: control or without isolate,
RE-2, FiE-3, FiE-2, MCE-1, FE-1, FE-2, SE-2, SE-3, and SE-S1.

2.1.2. Biochemical Test

• IAA Phytohormone Production Test
The ability of rhizobacteria to produce indole-3-acetic acid (IAA) was tested by inocu-
lating the rhizobacterial isolates into tryptic soy broth (TSB) medium. The isolates were
prepared as a 10 mL suspension with a cell density of 108 colony forming unit (CFU/mL−1 )
and incubated for 24 h. Then, a 3 mL aliquot of the bacterial suspension was transferred
into 30 mL of liquid TSB medium supplemented with tryptophan (1 mg.mL−1 ) and incu-
bated at 28 ◦ C on a shaker for six days [14]. To determine the concentration of IAA, 5 mL
of the supernatant with 2 mL of Salkowski reagent (1 mL 0.5 mM FeCl3 and 50 mL 35%
HClO4 ) [23,24]. The absorbance was then measured at a wavelength of 535 nm using a
spectrophotometer. IAA concentrations were determined with an IAA standard curve and
sterile medium as a blank.
• Organic Acid Production Test
High-performance liquid chromatography (HPLC) (Alliance® HPLC—e2695 Separa-
tions Module by Waters), the company is headquartered in Milford, MA, USA, was used
for the testing of organic acid production. The PGPR isolates were cultured in Okon’s
medium for 3 days. Standard organic acids with a concentration of 100 ppm were weighed
at 0.1 g each. The mobile phase consisted of KH2 PO4 with a concentration of 6.8 g and
pH of 2.8. The PGPR isolates samples were filtered through a 0.45 µm syringe filter into
1.5 mL vial bottles. The samples were then injected into a reverse-phase HPLC column
(Grace-smart RP 18.5 µm) and read at λ = 210 nm. The flow rate of the mobile phase was
set at 0.7 mL/min with an injection time of 10 min [25].
• Nitrogenase Activity Test
The testing of nitrogenase activity of isolates was carried out qualitatively using the
most probable number (MPN) method [26], which involved culturing the bacteria on semi-
solid selective NFb medium. The procedure began with preparing an isolated suspension,
which was then inoculated into test tubes containing 5 mL of nitrogen fixation bacteria
(NFb) medium. To serve as an indicator, bromothymol blue was added to the medium. The
test tubes were then incubated at room temperature for 24 h, and the observed changes
included the formation of a white pellicle and blue color in the medium.

2.2. Selection of Capable Isolates

The three capable isolates were selected using a scoring method, where nine isolates
were ranked based on their performance in enhancing rice growth characteristics, IAA
phytohormone test, qualitative test of nitrogenase activity, and organic acid production test.
Agriculture 2023, 13, 1987 4 of 21

The isolates were ranked from highest to lowest, and the three isolates with the highest
scores were chosen as the capable isolates.

2.3. Biomolecular Test

Biomolecular tests were conducted on the three capable isolates to determine their
species. The identification of PGPR isolates was performed through 16S rRNA gene se-
quencing using the oligonucleotide primers 27F (5’-AGAGTTTTGATCCTGGCTCAG-3’),
785F (5’-GGATTAGATACCCTGGTA-3’), and 1492R (5’-GGTTACCTTGTTACGACTT-3’)
(1st base). The sequence data were aligned with a system software aligner and analyzed
to identify the bacterium and its closest neighbors using the basic local alignment search
tool (BLAST) and then adjusted to the National Center for Biological Information (NCBI)
database. The partial 16 S rRNA gene sequences were deposited in the GenBank database.
Phylogenetic and molecular evolutionary analysis of the 16 S rDNA sequences were con-
ducted using MEGA4 software [21], and aligned using CLUSTAL-X [27]. The pairwise
evolutionary distance matrix was generated and the evolutionary tree was inferred using
the neighbor-joining method. The bootstrap test was performed to cluster together the
associated taxa. The evolutionary distances were compared using the maximum composite
likelihood method.

2.4. Application of Liquid Inoculant in Upland Rice Cultivation

2.4.1. Biofertilizer Formulation
The formulation of the PGPR (plant growth-promoting rhizobacteria) biofertilizer
inoculant involves using a liquid carrier [28] which comprises the following components:
3% glycerol, 3% molasses, 1% potassium sorbate, 1% Tween-20, and enriched with 1%
nutrients. The PGPR inoculant with a population density of 108 CFU.mL−1 is rejuvenated
in Okon’s liquid medium for 48 h. All carriers and nutrients were mixed and then sterilized
using an autoclave at a temperature of 121 °C and 15 Psi. The bacterial cell suspension is
added to the carrier in 1:9 ratio by injection and stored at room temperature, followed by
an incubation period of 4 months.

2.4.2. Field Pot Experiment

• Location and Experiment Design
The research was conducted at the Faculty of Agriculture, Padjadjaran University from
July to November 2022. The pot experiment was conducted to evaluate the application of
liquid biofertilizer using Jatinangor Inceptisol soil (Table 1) at a depth of 0–30 cm, which
has been dried, pulverized, and composited. The experiment was arranged as a factorial
randomized block design with three replications. The liquid biofertilizers (LB) consisted
of 4 levels, namely, control, seed treatment (ST) = 400 mL of LB kg−1 , soil application
(SA) = 10 L of LB ha−1 , and ST + SA = 400 mL of LB + 10 L of LB ha−1 ), while the N
fertilizers consisted of 4 levels (0, 50, 100 and 150 kg N ha−1 ).
• Planting Medium Preparation
A total of 10 kg of planting media passed through a 5 mm sieve from the top layer
(0–20 cm) of Inceptisols was placed into polybags measuring 45 × 45 cm. Around 5 cm
from the bottom, holes with a diameter of 3 mm were made to prevent waterlogging during
rainfall. One day before planting the Situ Bagendit rice seeds, water was added to the
planting media until it reached 80% of field capacity by adding 250 mL of water per kg of
soil media. Seed treatment was carried out by soaking 1 kg of seeds in 1 L of LB solution
for 1 h. Afterward, 3–4 seeds were placed into each hole (3–5 cm) and covered with soil.
The soil application treatment was performed by dissolving 10 L of LB solution with a
concentration of 5 cc per liter of water. Then, 5 mL of this solution was irrigated into
each planting hole containing 3–4 rice seeds per planting hole. Nitrogen (N) fertilizer was
applied three times (one third of the dose each time) on days 14, 28, and 42 after planting.
The basal fertilizer used phosphorus (P) with a dose of 46 kg P2 O5 per hectare (applied
Agriculture 2023, 13, 1987 5 of 21

once at the beginning of planting), and potassium (K) with a dose of 60 kg K2 O per hectare
was applied twice on days 14 and 42 days after planting by providing it in a band around
the plants. Subsequently, the polybag plants were placed in an open field with a planting
distance of 30 × 25 cm. Daily checks and water additions were done to maintain the soil
moisture at field capacity (moist soil). In addition, routine maintenance was performed to
manually remove weeds and control pests and diseases. Furthermore, nets were installed
to prevent bird attacks. Harvesting was carried out after the rice ripened, indicated by 80%
of the leaves turning yellow. Plant maintenance included daily watering, manual weed
removal, and manual pest and disease control. Harvesting was conducted when more than
80% of the grains were matured, as indicated by yellowing rice leaves.

Table 1. The chemical–physical–biological soil properties of Inceptisols in Jatinangor.

No. Parameter Unit Result Criteria

1. pH:H2 O - 5.52 Acid
2. Organic Matter (%) 1.52 Low
3. Nitrogen (%) 0.14 Low
4. C/N - 10.87 Moderate
5. Total Phosphorus (mg 100 g−1 ) 49.08 Hight
6. Available Phosphorus (Bray) (ppm P) 0.06 Very Low
7. Potassium (mg 100 g−1 ) 40.22 Moderate
8. Cation Exchange Capacity (CEC) (cmol kg−1 ) 13.66 -
Texture:
Sand (%) 5
9. Clay
Dust (%) 26
Clay (%) 69
Microbial Activity of Nitrogen-Fixing
10. CFU g−1 105 -
Bacteria

2.5. Parameters and Data Analysis

The growth characteristics analyzed in this study include the population of nitrogen-
fixing bacteria, N uptake, total N in soil, plant height, root length, number of tillers,
number of panicles per clump, 1000-grain weight, and grain yield. The relative agronomic
effectiveness (RAE) is calculated according to the following formula:

Tested bio f ertilizer yield − control

RAE = × 100% (1)
Standard bio f ertilizer yield − control

The observation data were analyzed using a factorial simple randomized block design
(RBD) at a significance level of 5% to determine the differences among the tested treat-
ments [29]. Subsequently, the relationship model between the yield of upland rice and the
observed variables was examined. The growth data, soil chemical and biological properties,
and yield were subjected to Pearson’s correlation analysis to determine the presence or
absence of relationships among the variables. Variables that showed significant correlations
were further analyzed using multiple linear regression analysis to identify the variables
that had the most significant impact on the yield components of upland rice. IBM SPSS
Statistics 26 was the software employed for performing statistical data analysis, and the
results of the regression analysis served as a reference for conducting path analysis, which
was performed using IBM SPSS AMOS version 26.

3. Results
3.1. Bioassay and Biochemical Test
3.1.1. Bioassay (Biological Assay)
The observations of the shoot height and root length showed different responses to
the addition of N-fixing PGPR isolates. The plant height was significantly affected by the
addition of these isolates, but there was no significant effect on the root length (Table 2). The
Agriculture 2023, 13, 1987 6 of 21

analysis of variance in Table 2 indicates that the isolates RE-2, FE-3, and FE-2 had different
shoot heights compared to the control. Isolate RE-2 exhibited a significantly higher shoot
height, showing a 209% increase compared to the control. This considerable increase in the
rice shoot growth is believed to be attributed to the influence of phytohormone produced
by the N-fixing bacterial isolate. Plant growth can be enhanced through the synthesis of
compounds that aid in nutrient uptake from the environment, one of which is indole-3-
acetic acid (IAA).

Table 2. Effects of PGPR Nitrogen-Fixing Isolates on Shoot Height, Root Length, Shoot Dry Weight,
Root Dry Weight.

28 Days after Planting

Isolate Code Shoot Height Root Length Shoot Dry Root Dry
(cm) (cm) Weight (mg) Weight (mg)
Control 7.37 ± 3.81 a 7.12 ± 4.81 11.15 ± 6.81 a 3.63 ± 1.81 a
RE-2 22.80 ± 10.91 b 7.95 ± 5.51 20.32 ± 5.91 c 8.76 ± 3.91 c
FiE-3 22.56 ± 9.87 b 7.31 ± 3.87 21.45 ± 8.87 c 8.73 ± 3.87 c
FiE-2 22.80 ± 8.53 b 7.76 ± 5.02 18.70 ± 4.07 b 6.21 ± 3.02 ab
MCE-1 19.49 ± 7.11 ab 7.82 ± 5.11 18.76 ± 4.11 b 9.31 ± 4.11 c
FE-1 13.76 ± 6.07 ab 7.80 ± 3.07 9.41 ± 3.02 a 7.13 ± 3.07 bc
FE-2 17.07 ± 8.18 ab 7.52 ± 6.18 11.80 ± 6.18 ab 5.71 ± 2.18 ab
SE-2 7.96 ± 3.02 a 7.14 ± 7.53 9. 36 ± 4.53 a 6. 31 ± 2.33 bc
SE-3 12.57 ± 5.07 ab 7.68 ± 5.07 12.57 ± 5.07 ab 7.68 ± 3.07 bc
SE-1 12.57 ± 6.74 ab 7.47 ± 6.74 12.57 ± 6.74 ab 7.47 ± 3.74 bc
Note: the average value followed by the same letter was not significantly different according to Duncan’s follow-up
test at the 0.05 significance level.

The change in the root length did not exhibit significant differences among all treat-
ments, but it did show variations in root morphology, which could have a greater impact on
root dry weight. The addition of the PGPR isolates had a significant effect on the shoot dry
weight and root dry weight of the rice. The analysis of variance revealed that the isolates
RE-2 and FiE-3 did not significantly differ in the shoot dry weight, but they both signifi-
cantly differed from the other treatments, displaying the most optimum shoot dry weight.
This result can be attributed to the fact that these two isolates also promoted good shoot
height. The root dry weight of all the treatments differed significantly from the control,
except for the isolates FE-2 and FiE-2. The root dry weight showed better and significant
results in the isolate inoculation treatments compared with the control. High levels of the
IAA hormone can regulate numerous physiological processes, including cell enlargement,
and can stimulate the development of lateral roots and root hairs, thus increasing plant dry
weight [30].

3.1.2. Biochemical
• IAA Phytohormone Production
The experimental results revealed that all nine isolates demonstrated the ability to
produce the phytohormone indole-3-acetic acid (IAA), as presented in Table 3. The biosyn-
thesis of IAA in bacteria involves tryptophan, which serves as the primary precursor
compound in various metabolic pathways. Tryptophan is commonly recognized as a
crucial component in IAA formation, as the addition of tryptophan to bacterial cultures
stimulates enhanced IAA synthesis. The production of IAA by Gram-negative bacteria with
tryptophan follows the following pathways: (1) the indole-3-acetamide (IAM) pathway,
(2) the indole-3-pyruvic acid (IPyA) pathway, (3) the tryptophan chain oxidation pathway,
(4) the tryptamine-TAM pathway, and (5) the indole-3-acetonitrile pathway [31].
 Agriculture 2023, 13, 1987 7 of 21

Table 3. Ability of PGPR Isolates to Produce IAA Growth Hormones and Organic Acids.

28 Days after Planting

Organic Acids (ppm)
Isolate Code IAA (ppm)
Citrate Acetate Oxalate Lactate Malate Ascorbate
RE-2 4.97 8.074 6.019 3.909 4.118 nd 2.065
FiE-3 5.09 nd nd nd 1.087 2.394 nd
FiE-2 4.67 nd 5.925 1.150 nd nd nd
MCE-1 4.93 nd nd nd 2.801 8.192 nd
FE-1 4.69 nd nd nd nd 1.100 nd
FE-2 3.93 nd nd nd nd 1.218 nd
SE-2 4.56 nd nd nd nd 2.077 nd
SE-3 4.57 nd nd nd 9.876 5.077 nd
SE-1 5.30 nd nd 5.545 1.314 8.963 nd
Notes: nd: not identified.

• Organic Acid
The results of the organic acid production assay on the nine PGPR isolates revealed
that all the isolates demonstrated the capability to produce various types and quantities
of organic acids. According to the analysis presented in Table 3, it was observed that
isolate FiE-3 exhibited the highest diversity of organic acid production among all the PGPR
isolates tested. This isolate was capable of synthesizing multiple organic acids, including
citrate, acetate, oxalate, lactate, and ascorbate. On the other hand, the isolates FE-2, SE-1,
and SE-2 showed a more limited capacity for organic acid production, with each of them
only capable of producing a single organic acid, which in this case was malic acid. The
ability of the microbes to produce organic acids is also worth evaluating because organic
acids function to dissolve minerals in the soil, making them available to plants. Plant
growth-promoting rhizobacteria can directly or indirectly contribute to the plant growth.
This bacterial strain produces cytokinins, auxin, and ACC-deaminase, while also releasing
organic acids. The release of these organic acids plays a crucial role in facilitating the plant
nutrient uptake. Organic acids such as malonate, oxalate, glycolate, acetate, and formic
acid contribute to the acquisition of essential elements such as phosphorus, calcium, iron,
zinc, and manganese in soils with limited nutrient availability [32].
• Nitrogenase Activity
Another functional characteristic that was tested was the ability of the isolates to pro-
duce the nitrogenase enzyme in nitrogen-free semi-solid media, specifically NFb medium.
Agriculture 2023, 13, x FOR PEER REVIEW 8 of 22
The capacity of the bacteria to fix nitrogen can be observed by the formation of a white
pellicle on the media and changes in the color of the NFb medium (Figure 1).

White rings

Figure 1. 1.Qualitative
Figure Qualitative Nitrogenase ActivityAssessed
Nitrogenase Activity Assessed by Formation
by Formation of White
of White Rings
Rings on on the
the NFb NFb
Media.
Media.
The formation of the white rings is caused by the absence of oxygen in the media,
while
Thethe color change
formation in white
of the the NFb medium
rings occursby
is caused due
thetoabsence
the bromothymol
of oxygenblue indicator
in the media,
shifting
while to blue
the color at higher
change in thepH
NFblevels, indicating
medium occursthe
duenitrogenase activity [33].
to the bromothymol Moreover,
blue indicator
shifting to blue at higher pH levels, indicating the nitrogenase activity [33]. Moreover, the
most probable number (MPN) method was employed to estimate the population of
nitrogen-fixing microorganisms. The results of the population estimation for
nitrogen-fixing microorganisms are presented in Table 4.
Agriculture 2023, 13, 1987 8 of 21

the most probable number (MPN) method was employed to estimate the population of
nitrogen-fixing microorganisms. The results of the population estimation for nitrogen-fixing
microorganisms are presented in Table 4.

Table 4. Estimation of Nitrogen-Fixing Microorganisms using the Most Probable Number (MPN)
Method.

Isolate Code MPN

CFU mL−1
10−1 10−2 10−3
RE-2 3 3 2 1.1 × 105
FiE-3 3 2 1 1.5 × 104
FiE-2 3 2 1 1.5 × 104
MCE-1 3 3 3 1.1 × 105
FE-1 3 3 3 1.1 × 105
FE-2 3 2 0 9.3 × 103
SE-2 3 3 3 1.1 × 105
SE-3 3 3 3 1.1 × 105
SE-1 3 2 0 9.3 × 103

3.2. The Selection of Capable Isolates

The identification of the promising isolates was carried out through a scoring system
based on the results of the bioassay, IAA production test, organic acid test, and qualitative
nitrogenase activity test. The isolates were ranked according to their scores, with the
highest-scoring isolate being ranked first, followed by the second and third ranks.
Table 5 presents the ranking results, revealing that isolate RE-2 achieved the highest
score and obtained the first rank, followed by isolate MCE-1 in the second rank, and isolate
FE-3 in the third rank. Notably, isolate RE-2 was originally isolated from a rainfed field
ecosystem with upland rice vegetation, while isolate MCE-1 was obtained from a mixed
crop ecosystem with cashew vegetation, and isolate FE-3 originated from a field ecosystem
with maize vegetation. These three top-ranked isolates are considered capable isolates.
Subsequently, the molecular testing was conducted to determine the species names of these
isolates, and they were further utilized in the subsequent experiment on the upland rice.

Table 5. Isolate Ranking (Scoring) Based on Plant Height (PH), Root Length (RL), Shoot Dry Weight
(SDW), Root Dry Weight (RDW), Nitrogenase (N).

Biological Test
Isolate Code Score Total Ranking
PH RL SDW RDW IAA Org. Acid N
RE-2 8 9 8 8 2 8 9 52 1
FiE-3 7 2 9 7 3 9 8 45 3
FiE-2 9 6 6 3 7 4 8 43 4
MCE-1 6 8 7 9 4 5 9 48 2
FE-1 4 7 2 4 6 6 9 38 6
FE-2 5 4 3 1 5 1 7 26 8
SE-2 1 1 1 2 1 2 9 17 9
SE-3 2 5 4 6 8 3 9 37 7
SE-1 3 3 5 5 9 7 7 39 5

3.3. Biomolecular Test Results

The species lineage of the three isolates is depicted in their respective phylogenetic
trees shown in Figure 2. A phylogenetic tree is a diagram that visualizes the evolutionary
relationships and origins of microorganisms, leading to the formation of species with a
similarity above 99% [34]. According to the phylogenetic tree analysis, isolate RE-2 was
found to be derived from Bacillus pumilus strain PLK3, undergoing gradual evolution while
retaining certain similarities until it eventually forms the final species, Bacillus sp. On the
Agriculture 2023, 13, 1987 9 of 21

other hand, the isolates MCE-1 and FiE-3 trace their origins back to different strains of
Agriculture 2023, 13, x FOR PEER REVIEW 10 of 22
Bacterium and subsequently evolve into their respective final species Delftia tsuruhatensis
(Figure 2).

Figure 2. A Phylogenetic Tree of Isolates MCE-1, RE-2, and FiE-3.

Figure 2. A Phylogenetic Tree of Isolates MCE-1, RE-2, and FiE-3.
A
A phylogenetic
phylogenetic tree
tree is
is aa diagram
diagram that
that describes
describes the
the origin
origin and
and evolution
evolution ofof microbes.
microbes.
Bacteria belonging to the genus Delftia have been extensively isolated
Bacteria belonging to the genus Delftia have been extensively isolated from various from various
en-
environments including seawater, soil, and plants. These bacteria have
vironments including seawater, soil, and plants. These bacteria have been extensively been extensively
studied
studied for
for their
their capacity
capacity toto degrade
degrade specific
specific organic pollutants, including
organic pollutants, including chloroanilines
chloroanilines
and
and heavy
heavy metals
metals like
like chromium.
chromium. Recent
Recent research
research has
has revealed
revealed that
that this
this bacterial
bacterial genus
genus isis
also classifiedasas
also classified a plant
a plant growth-promoting
growth-promoting bacteria.
bacteria. Their
Their role role growth
as plant as plant growth
promoters
promoters
contributes contributes
to enhancedto enhanced
plant growthplant growth by
by facilitating facilitating
nutrient nutrient
availability availability
to the to
host plants,
the host plants, reducing nitrogen, producing organic acids, and providing
reducing nitrogen, producing organic acids, and providing protection against pathogenic protection
against pathogenic infections [35]. On the other hand, Bacillus sp. bacteria belong to a
group of rhizobacteria that function as plant growth promoters through nitrogen
fixation, without forming symbiotic relationships with plants. These bacteria possess
Agriculture 2023, 13, 1987 10 of 21

Agriculture 2023, 13, x FOR PEER REVIEW 11 of 22

infections [35]. On the other hand, Bacillus sp. bacteria belong to a group of rhizobacteria
that function as plant growth promoters through nitrogen fixation, without forming symbi-
otic relationships
various withcharacteristics
functional plants. These bacteria possess their
that highlight various functional
ability characteristics
as biological agentsthat
for
highlight their ability
promoting plant growth. as biological agents for promoting plant growth.
Molecular
Molecularidentification
identificationofofthe
thethree
threecapable
capableisolates
isolateswas
wasperformed
performedby bydata
dataanalysis
analysis
using
using BioEdit software version 7.0.5.3, which generated the lengths of the 16S rRNAgene
BioEdit software version 7.0.5.3, which generated the lengths of the 16S rRNA gene
sequences
sequencesfor forisolate FiE-3
isolate as as
FiE-3 614614
bp,bp,
isolate MCE-1
isolate MCE-1as 822 bp, and
as 822 isolate
bp, and RE-2RE-2
isolate as 1388
as bp).
1388
The molecular results for the three isolates are presented in Table 6 and Figure 3.
bp). The molecular results for the three isolates are presented in Table 6 and Figure 3.

Table6.6.Results
Table ResultsofofBlast-n
Blast-nAnalysis
Analysisofofthe
the16S
16SrRNA
rRNASequences
SequencesofofIsolates
IsolatesB3,
B3,C1,
C1,and
andA2.
A2.
Isolate
Isolate Code SpeciesSpecies
Code Code Code Species
Species NameName Homology
Homology(%)(%)
FiE-3
FiE-3 DS 9 DS 9 Delftia
Delftia tsuruhatensis
tsuruhatensis strain
strain D9D9 99.84
99.84
MCE-1
MCE-1 DS DS Delftia sp. strain
Delftia sp. strain MS2As2MS2As2 99.64
99.64
RE-2 BS Bacillus sp. 99.33
RE-2 BS Bacillus sp. 99.33

Figure3.3.Electrophoretic
Figure Electrophoreticanalysis
analysisofofPCR.
PCR.

3.4.
3.4.Total
TotalNitrogen
NitrogenandandNitrogen
NitrogenUptake
Uptake
According to Duncan’s multiple
According to Duncan’s multiple range rangetest,
test,treatment
treatmentc3nc32 n(seed-soil
2 (seed-soil application
application 10 L
10haL−1ha −1 + 100 kg ha−1 N dosage) exhibited the highest total nitrogen content at 0.31%
+ 100 kg ha N dosage) exhibited the highest total nitrogen content at 0.31% (Table
−1
(Table 7). represented
7). This This represented an increased
an increased soil soil nitrogen
nitrogen totaltotal compared
compared to to
thethe initialvalue
initial valueof
of0.14%.
0.14%.This
Thistreatment
treatmentalsoalsoincreases
increasesthethesoil
soil nitrogen
nitrogen content
content by by up
up to a 21.05% riseinin
to a 21.05% rise
total
totalnitrogen
nitrogencompared
comparedtotothe thetreatment
treatmentwithout
withoutliquid
liquidbiofertilizer
biofertilizeratatthe
thesame
samenitrogen
nitrogen
dosage.
dosage. These findings suggest that the application of a liquid biofertilizer contributestoto
These findings suggest that the application of a liquid biofertilizer contributes
the
theenhanced
enhancednitrogen
nitrogenavailability
availabilityininthe
thesoil.
soil.
This
This observation aligns with the studyconducted
observation aligns with the study conductedby by[30]
[30]which
whichdemonstrated
demonstratedthat that
the addition of biofertilizer to chemical fertilizers significantly increased the total nitrogen
the addition of biofertilizer to chemical fertilizers significantly increased the total
content compared to the treatments without biofertilizer. Additionally, the total nitrogen
nitrogen content compared to the treatments without biofertilizer. Additionally, the total
content in the treatment using 100% of the recommended chemical fertilizer dosage was
nitrogen content in the treatment using 100% of the recommended chemical fertilizer
comparable to that of the soil in the treatment employing 50% of the nitrogen dosage along
dosage was comparable to that of the soil in the treatment employing 50% of the nitrogen
with biofertilizer supplementation [36].
dosage along with biofertilizer supplementation [36].
The analysis of variance revealed a significant influence of the combined treatment
The analysis of variance revealed a significant influence of the combined treatment
of liquid biofertilizer and nitrogen dosage on the nitrogen uptake of plants. The uptake
of liquid biofertilizer and nitrogen dosage on the nitrogen uptake of plants. The uptake of
of nitrogen by plants is influenced by their nitrogen requirements and the availability of
nitrogen by plants is influenced by their nitrogen requirements and the availability of
nitrogen in the soil. When nitrogen is abundantly available, its concentration in plant
nitrogen in the soil. When nitrogen is abundantly available, its concentration in plant
tissues is high (approximately 4.5%) and gradually decreases as the plants mature.
tissues is high (approximately 4.5%) and gradually decreases as the plants mature.
The combination of the liquid biofertilizer treatment and nitrogen dosage has been
proven to effectively meet the nitrogen nutrient requirements of rice plants. According
Table 7. The Effect of Liquid Biofertilizer as Seed Treatment (ST) or Soil Application (SA) and
to [35] the nitrogen content in rice plants is considered adequate within the range of
Nitrogen Dosage on Total Nitrogen and Nitrogen Uptake at Maximum Vegetative (63 Days after
Planting).

LB Application (C) Nitrogen Fertilizers (N) (kg ha−1)

Agriculture 2023, 13, 1987 11 of 21

2.6–3.20%. Other studies suggest that the critical nitrogen level for many plants is below
3% [37]. The research findings indicate that all treatments resulted in high nitrogen uptake
by the rice plants, with the c1 n2 (seed treatment 400 mL kg− 1 seed + 100 kg ha− 1 nitrogen
dosage) treatment exhibiting the highest percentage of nitrogen uptake at 4.50% (Table 7).
This can be attributed to the fact that the microbial-based fertilizers offer optimal nitrogen
availability during the vegetative growth stage [35].

Table 7. The Effect of Liquid Biofertilizer as Seed Treatment (ST) or Soil Application (SA) and Nitrogen
Dosage on Total Nitrogen and Nitrogen Uptake at Maximum Vegetative (63 Days after Planting).

Nitrogen Fertilizers (N) (kg ha−1 )

LB Application (C)
n0 (0) n1 (50) n2 (100) n3 (150)
Total Nitrogen (%) ± SD
c0 = control 0.12 ± 0.01 aA 0.21 ± 0.01 bB 0.20 ± 0.01 aB 0.19 ± 0.01 aB
c1 = 400 mL kg−1 seed ST 0.19 ± 0.01 bA 0.20 ± 0.00 aA 0.22 ± 0.01 bB 0.22 ± 0.01 bB
c2 = 10 L ha−1 SA 0.19 ± 0.00 bA 0.20 ± 0.00 aB 0.22 ± 0.01 bB 0.23 ± 0.01 bB
c3 = c1 + c2 0.18 ± 0.01 bA 0.21 ± 0.00 bB 0.31 ± 0.01 cC 0.20 ± 0.01 bB
Nitrogen Uptake (%) ± SD
c0 = control 3.28 ± 1.45 aA 3.64 ± 1.75 aB 4.16 ± 2.71 cC 4.14 ± 2.77 cC
c1 = 400 mL kg−1 seed ST 3.48 ± 1.56 aA 3.86 ± 2.99 cC 3.84 ± 2.87 bC 4.25 ± 2.62 dD
c2 = 10 L ha−1 SA 3.36 ± 1.75 aA 3.93 ± 2.38 dB 4.50 ± 2.54 dD 4.16 ± 2.73 cC
c3 = c1 + c2 3.37 ± 1.36 aA 3.73 ± 2.63 bB 3.72 ± 2.69 bB 3.92 ± 1.39 bC
Nitrogen Uptake (mg kg−1
DM)
± SD
c0 = control 74.99 ± 0.45 aB 84.19 ± 0.75 aC 64.29 ± 0.71 aA 64.43 ± 0.54 aA
c1 = 400 mL kg−1 seed ST 88.26 ± 0.56 cA 98.45 ± 0.99 dC 100.87 ± 0.87 cD 96.06 ± 1.62 dB
c2 = 10 L ha−1 SA 80.56 ± 0.75 bA 89.28 ± 0.38 bC 82.39 ± 0.77 cB 89.98 ± 0.73 cC
c3 = c1 + c2 83.35 ± 0.39 cB 83.01 ± 0.63 aB 70.14 ± 0.69 bA 88.56 ± 0.36 cC
The mean value followed by the same letter was not significantly different according to Duncan’s follow-up test
at the 0.05 significance level. Lowercase letters are read vertically. Capital letters are read horizontally.

This result can be interpreted as the application of the biofertilizer stimulating an

increase in the number of microbes, which consequently enhances the nitrogen content in
the soil available for plant absorption. Another opinion supports this idea by suggesting
that the nitrogen content in the soil is closely related to the number of microbial populations
in the soil so that it helps plants absorb N [25].

3.5. Nitrogen-Fixing Bacteria (NFB) Population

The combination of the liquid biofertilizer treatment and nitrogen dosage significantly
affected and interacted with the response of the tested nitrogen-fixing bacteria (NFB)
population. In general, the use of a liquid biofertilizer increased the population of the NFB
in the research soil.
The initial soil had an NFB population of 105 CFU g−1 , which increased in all treat-
ments to 1 × 108 CFU g−1 , except for the control. The population of the NFB increased with
the application of the liquid biofertilizer, whether through seed treatment, soil application,
or seed-soil application, with a 150 kg ha− 1 N dosage. Treatment c3 n3 (seed-soil application
10 L.ha−1 + 150 kg ha−1 N dosage) was the best treatment in maintaining the NFB popu-
lation, with a population of 8.20 × 108 CFU g− 1 (Table 8). The NFB population increased
with the increase in liquid biofertilizer, both with the application of the seed treatment, soil
application and seed-soil application of 10 L ha− 1 with 150 kg ha−1 N dosage. The large
population of the NFB in this treatment made it possible to provide the liquid biofertilizer
with two applications at once, namely, in the seeds and leaking it into the soil with sufficient
doses of nitrogen as a source of nutrition for the microbes and plants.
Agriculture 2023, 13, 1987 12 of 21

Table 8. The Effect of Liquid Biofertilizer as Seed Treatment (ST) or Soil Application (SA) and
Nitrogen Dosage on Population of N-Fixing Bacteria.

Nitrogen Fertilizers (N) (kg·ha−1 )

LB Application (C)
n0 (0) n1 (50) n2 (100) n3 (150)
NFB Population × 108 (CFU g−1 ) ± SD
c0 = control 0.83 ± 0.35 aA 2.53 ± 1.56 aB 1.16 ± 0.45 aA 0.86 ± 0.41 aA
c1 = 400 mL kg−1 seed ST 1.13 ± 0.15 aA 2.96 ± 0.11 aB 1.13 ± 0.45 aA 5.06 ± 0.70 bC
c2 = 10 L ha−1 SA 1.70 ± 0.95 aA 3.20 ± 1.20 abB 5.33 ± 1.10 bC 7.67 ± 0.28 aA
c3 = c1 + c2 1.50 ± 0.17 aA 4.10 ± 1.01 bB 1.70 ± 0.20 aA 8.20 ± 1.01 cC
The mean value followed by the same letter was not significantly different according to Duncan’s follow-up test
at the 0.05 significance level. Lowercase letters are read vertically. Capital letters are read horizontally.

3.6. Liquid Biofertilizer Inoculant Application on Upland Rice

The combination of the liquid biofertilizer treatment with the nitrogen (N) dosage did
not exhibit a significant interaction in terms of the plant height response (Table 9). This
could be attributed to the fact that the plant height of the rice is primarily influenced by the
genetic factors inherent to each variety. In this study, the same variety, Situ Bagendit, was
used in addition to the environmental factors.

Table 9. Effect of Liquid Biofertilizer as Seed Treatment (ST) or Soil Application (SA) and Nitrogen
Dosage on Plant Height, Number of Tillers and Root Length at Maximum Vegetative (63 Days
after Planting).

Nitrogen Fertilizers (N) (kg·ha−1 )

LB Application (C)
n0 (0) n1 (50) n2 (100) n3 (150)
Plant Height (cm) ± SD
c0 = control 37.69 ± 3.35 47.37 ± 4.58 47.37 ± 4.58 45.21 ± 5.47
c1 = 400 mL kg−1 seed ST 46.53 ± 4.87 47.37 ± 4.58 47.37 ± 4.58 45.21 ± 5.47
c2 = 10 L ha−1 SA 47.32 ± 3.43 45.21 ± 5.47 47.37 ± 4.58 45.21 ± 5.47
c3 = c1 + c2 47.32 ± 3.43 48.67 ± 4.56 47.37 ± 4.58 45.21 ± 5.47
Number of Tillers ± SD
c0 = control 19.67 ± 6.81 aB 21.67 ± 4.51 aA 21.33 ± 2.52 aA 18.00 ± 4.58 aA
c1 = 400 mL kg−1 seed ST 21.67 ± 2.08 aA 23.67 ± 4.93 aA 27.00 ± 4.36 bB 21.56 ± 1.50 aA
c2 = 10 L ha−1 SA 22.33 ± 6.66 aA 32.67 ± 3.51 bB 23.67 ± 5.03 aA 29.00 ± 3.00 bAB
c3 = c1 + c2 22.00 ± 4.36 aA 20.33 ± 1.53 aA 24.33 ± 3.21 aA 24.33 ± 1.15 aA
Root Length (cm) ± SD
c0 = control 27.17 ± 1.76 aA 28.76 ± 2.40 abAB 26.67 ± 2.08 aA 27.50 ± 2.29 aAB
c1 = 400 mL kg−1 seed ST 26.52 ± 2.64 aA 28.50 ± 0.50 abA 28.33 ± 1.53 aA 29.21 ± 2.07 aA
c2 = 10 L ha−1 SA 29.17 ± 2.36 aA 26.54 ± 1.28 aA 29.67 ± 2.52 aA 28.50 ± 1.50 aA
c3 = c1 + c2 30.23 ± 4.77 aA 30.53 ± 3.10 bA 36.10 ± 3.48 bB 28.83 ± 2.02 aA
The mean value followed by the same letter was not significantly different according to Duncan’s follow-up test
at the 0.05 significance level. Lowercase letters are read vertically. Capital letters are read horizontally.

The plant height in the control treatment was the lowest, suggesting a limited avail-
ability of nutrients for promoting the plant growth. The presence of the nitrogen-fixing
PGPR (plant growth-promoting rhizobacteria) in the biofertilizer and their production
of IAA can stimulate root development, ultimately leading to an increased plant height.
Plant growth-promoting rhizobacteria play a main role in enhancing the rice growth by
producing phytohormones, particularly IAA, which positively influence the biomass, root
branching, and nitrogen content of plants. Additionally, the application of different nitrogen
doses contributes to the availability of nitrogen for plant uptake and utilization [38,39].
The application of the liquid biofertilizer at different levels of nitrogen dosage signifi-
cantly interacted and resulted in a significant difference in the number of tillers. Treatment
Agriculture 2023, 13, 1987 13 of 21

c2 n1 (soil application 10 L ha− 1 + 50 kg ha− 1 N) produced an average of 32.67 tillers. This

treatment showed an increase in the number of tillers by 66% compared to the control
treatment. These findings are consistent with the study conducted by [40], which showed
that the 100% N fertilizer treatment resulted in the same number of tillers as the biofertilizer
treatment with 75% N dosage. The application of a liquid biofertilizer effectively provides
nitrogen nutrients for rice. The availability of nitrogen in the rhizosphere determines plant
growth. The plant height and the number of tillers are morphological characteristics of rice
that impact the rice yield components. Plants require high levels of nitrogen during the
vegetative phase. The presence of a biofertilizer in the soil is highly effective in improving
the nutrient availability for plants.
The subsequent analysis demonstrated a significant effect of the liquid biofertilizer
application and nitrogen dosage on increasing the length of the rice roots. Treatment c3 n2
(seed-soil application of 10 L ha− 1 + 100 kg h− 1 nitrogen dosage) resulted in an average root
length of 36.10 cm. This treatment exhibited a 32.86% increase in the root length compared
with the control treatment. This enhancement is attributed to the presence of the PGPR
microbes in the liquid biofertilizer. When applied through a combination of seed soaking
and direct soil application, these microbes stimulate the production of the growth hormone
IAA, leading to improved root length. Root colonization by bacteria plays an important
role in plant growth and protects plants from pathogens in the soil. IAA-producing bacteria
inoculation can optimize the lateral and adventitious roots, root hair formation, and main
root elongation [37].

3.7. Yield Components

The combination of the liquid biofertilizer treatment with the nitrogen dose resulted in
a significant interaction that influenced the number of panicles. Further tests with Duncan’s
multiple range test with a significance level of 5% showed that the c2 n1 treatment (soil
application 10 L ha− 1 + 50 kg ha− 1 N dosage) had a panicle number of 29 stems (Table 10)
and this number is equal to the c2 n3 treatment (soil application 10 L ha− 1 + 150 kg ha− 1 N
dosage).
These results concluded that the application of the liquid biofertilizer could reduce
the dosage of inorganic fertilizer with the same results as higher nitrogen doses. Besides
being able to reduce the dose of nitrogen, the liquid biofertilizer administration was also
able to increase the number of panicles by 163.63% compared to the control treatment. The
number of grains represents the quantity of grains produced within a single hill per plant
pot. The application of a liquid biofertilizer with a nitrogen dosage resulted in a significant
interaction on the number of grains per hill. Further analysis using Duncan’s multiple
range test at a 5% significance level showed that treatment c2 n1 (soil application 10 L ha− 1
+ 50 kg ha− 1 N dosage) had a similar number of grains compared to treatment c2 n3 (soil
application 10 L ha− 1 + 150 kg ha− 1 N dosage), which was 1030 grains.
The application of the liquid biofertilizer significantly increased the number of grains
per hill by enhancing the yield by 115.93% compared to the treatment without liquid
biofertilizer at the same nitrogen fertilizer dosage. This is possible due to the role of
microorganisms present in the biofertilizer, which can fix N2 from the air and convert
it into a form available for plants to fulfill their nutritional requirements. The optimal
nutrient uptake can enhance flower formation and subsequently the grain production,
thus increasing the percentage of grains per hill. The number of grains per hill is closely
related to grain yield (Table 10). Observations of the number of grains indicate that the
application of the biofertilizer can increase the number of grains per hill in the rice plants.
All treatments with the addition of the biofertilizer resulted in a higher number of grains
per hill compared to the control. This finding is also supported by the study conducted
by [41] which showed that the use of biofertilizer increased the average number of grains
by 46.39% compared to the chemical fertilizer treatment. The application of biofertilizer,
which can fix nitrogen from the air, also needs to be supplemented with available inorganic
Agriculture 2023, 13, 1987 14 of 21

N and biologically available P. Therefore, the use of biofertilizer can enhance the growth of
rice and yield components [42].

Table 10. Effect of Liquid Biofertilizer as Seed Treatment (ST) or Soil Application (SA) and Nitrogen
Dosage on Yield Components.

Nitrogen Fertilizers (N) (kg ha−1 )

LB Application (C)
n0 (0) n1 (50) n2 (100) n3 (150)
Number of Panicles ± SD
c0 = control 11 ± 2 aA 18 ± 2 aB 18 ± 1 aB 20 ± 5 aB
c1 = 400 mL kg−1 seed ST 12 ± 1 aA 16 ± 3 aA 25 ± 1 bB 20 ± 4 aA
c2 = 10 L ha−1 SA 15 ± 1 aA 29 ± 3 bC 21 ± 6 abB 27 ± 3 bC
c3 = c1 + c2 12 ± 5 aA 16 ± 4 aAB 23 ± 6 abBC 21 ± 5 aB
Number of Grains ± SD
c0 = control 351 ± 8 aA 477 ± 27 aB 462 ± 10 aB 370 ± 5 aA
c1 = 400 mL kg−1 seed ST 429 ± 9 bA 688 ± 7 bB 789 ± 66 cC 989 ± 77 bD
c2 = 10 L ha−1 SA 457 ± 19 cA 1030 ± 64 dC 619 ± 98 bB 1054 ± 63 bcC
c3 = c1 + c2 387 ± 38 bcA 868 ± 31 cC 672 ± 60 bB 989 ± 27 cC
Weight of 1000 Grains (g) ± SD
c0 = control 19.62 ± 0.04 aA 26.16 ± 0.13 aAB 27.98 ± 0.12 aB 23.73 ± 0.16 aAB
c1 = 400 mL kg−1 seed ST 19.72 ± 0.09 aA 27.72 ± 0.07 aB 25.79 ± 0.12 aAB 24.03 ± 0.24 abAB
c2 = 10 L ha−1 SA 19.77 ± 0.09 aA 27.87 ± 0.24 aB 27.95 ± 0.18 aB 27.92 ± 0.16 bB
c3 = c1 + c2 20.69 ± 0.03 aA 27.85 ± 0.07 aB 26.99 ± 0.17 aAB 27.72 ± 0.13 bB
Grain Yields (g pot−1 ) ± SD
c0 = control 9.05 ± 0.54 bA 13.67 ± 1.36 aB 13.03 ± 0.46 aB 8.55 ± 0.11 aA
c1 = 400 mL kg−1 seed ST 5.70 ± 0.20 aA 18.75 ± 0.66 bB 21.33 ± 1.64 dC 26.64 ± 0.42 bD
c2 = 10 L ha−1 SA 11.60 ± 0.43 cA 29.81 ± 1.00 dC 15.45 ± 1.11 bB 30.01 ± 1.45 cC
c3 = c1 + c2 10.88 ± 0.78 cA 24.67 ± 1.24 cC 18.98 ± 1.50 cB 27.65 ± 1.69 cC
The mean value followed by the same letter was not significantly different according to Duncan’s follow-up test
at the 0.05 significance level. Lowercase letters are read vertically. Capital letters are read horizontally.

The application of a liquid biofertilizer combined with nitrogen dosage levels showed
significant interactions and results on the weight of 1000 grains. The analysis using Dun-
can’s multiple range test at a 5% significance level showed that treatment c2 n1 (soil applica-
tion 10 L ha− 1 + 50 kg ha− 1 nitrogen dosage) had a weight of 1000 grains which did not
differ from treatment c2 n3 (soil application 10 kg ha− 1 + 150 kg ha− 1 N dosage), which was
27.87 g (Table 10). The weight of 1000 grains obtained in this study corresponds to that of
the Situ Bagendit variety, which has a weight of 27.5 g for 1000 grains. The increase in the
yield components is also evident as an effect of the biofertilizer treatment. The increase in
the yield components occurred in terms of the number of grains per hill and the weight of
1000 grains.
Further test results showed that the addition of the liquid biofertilizer tended to
increase the grain yield when compared to the control treatment. The best effect of adding
liquid biofertilizer resulted from the liquid biofertilizer c2 n3 treatment (soil application
10 L ha−1 + 150 kg ha−1 N dosage) of 30.01 g pot−1 , but this result was not different from
the n2 c1 treatment (soil application 10 L ha−1 + 50 kg ha−1 N dosage) which is capable of
producing 29.81 g pot−1 grain yield which is equal to 3.98 ton ha−1 . These results indicate
that the addition of the liquid biofertilizer was able to reduce the nitrogen dose and increase
the grain yield by 102% compared to the treatment without liquid biofertilizer with the
same nitrogen dose. This is presumably because the activity of the nitrogen fixing bacteria
(NFB) in the liquid biofertilizer has the effect of increasing the nutrient uptake and nutrient
availability in the soil so that the yield component increases when the nitrogen fertilizers
are applied together with biofertilizer.
Agriculture 2023, 13, 1987 15 of 21

3.8. Relative Agronomic Effectiveness (RAE)

The effectiveness of the biological fertilizers in increasing the agronomic value of
plants can be seen from the relative agronomic effectiveness (RAE) value, specifically if the
RAE value is above 95%. It is calculated by dividing the difference between the yield of the
tested fertilizer and the control fertilizer by the difference between the yield of the standard
fertilizer and the control fertilizer, then multiplying the result by 100%.
Based on the calculation, the RAE value for the treatment of biofertilizer soil applica-
tion at a rate of 10 L ha−1 + 50 kg ha−1 N dosage was found at 238.08% (Table 11). This
RAE value demonstrates that the application of the biofertilizer has a significant positive
impact on enhancing the agronomy of the upland rice in this study because the RAE value
is above 95%. The effectiveness of a particular biological fertilizer is determined by the
microbial density of the biofertilizer, which contributes to the increased grain yield of the
crop and subsequently leads to a higher RAE value [43].

Table 11. RAE Values of Upland Rice Due to Liquid biofertilizers as Seed Treatments (ST) or Soil
Application (SA) and Nitrogen Fertilizers.

RAE (%)
Nitrogen (N) (kg ha−1 )
Biofertilizers (P)
N0 (0) N1 (50) N2 (100) N3 (150)
c 0 = control 0 46.52 100 −30.33
c 1 = 400 mL kg−1 seed ST −33.33 123.01 159.64 201.75
c 2 = 10 L ha−1 SA 32.45 235.08 138.59 238.59
c 3 = c1 + c2 23.68 22.43 163.02 228.07

3.9. Model of Relations between Variables on Grain Yield for Upland Rice
The correlation is technical analysis of measuring the associations or the strength of the
relationship between more than one variable. The strength of the relationship is measured
between values of −1 to +1. A correlation analysis is carried out if the classical conditions
are met, namely if the results of the treatment are interacting significantly. The treatment
of the liquid biological fertilizers and nitrogen doses interacted and significantly resulted
in a correlation with the following variables: the number of tillers, population of N-fixing
bacteria, N uptake, total N content of the soil, number of panicles, number of grains, weight
of 1000 grains, and grain yield. The correlation coefficient is positive, meaning that an
increase in one character will be followed by an increase in another character.
Conversely, a negative correlation coefficient means that an increase in one character
will decrease the other character. The level of correlation is divided based on the beta value
with the categories: β < 0.2 meaning a low correlation, β between 0.2 and 0.5 has a moderate
correlation, and β > 0.5 has a high correlation [44]. The correlation analysis revealed that
the variables of the N uptake, population of N-fixing bacteria, number of panicles, weight
of 1000 grains, and number of grains per clump exhibited a positive correlation with the
grain yield (Table 12). This suggests that an increase in these variables is associated with an
increase in the grain yield. The subsequent analysis involved a multiple linear regression
to assess the strength of the relationship between one or more independent variables and
the dependent variable (the grain yield).
The results of the multiple linear regression analysis demonstrate the significant
contributions of each independent variable, all of which exhibit a positive correlation.
The effective contributions of these variables to grain yield (Table 12) in descending
order from largest to smallest are as follows: the number of grains (87.27%), weight of
1000 grains (5.89%), number of panicles (1.85%), N-fixing bacteria population (1.2%),
and N uptake (1.16%).
The number of panicles represents the quantity of fruits or seeds produced in each
clump. A higher number of panicles indicates a greater potential for increased grain weight
due to a higher yield. The positive correlation between the density of the N-fixing bacteria
Agriculture 2023, 13, 1987 16 of 21

(NFB) and the N uptake with the grain weight can be attributed to the role of the N-fixing
bacteria population in enhancing the soil fertility and increasing the availability of nitrogen,
which is subsequently absorbed by plants [45]. The uptake of nitrogen has a direct impact
on the rice grain development, as an increase in the N uptake leads to an increase in the
grain yield [46].

Table 12. Multiple Linear Regression Results between Grain Yield (Y) and N Uptake (X1), Number
of Panicles (X2), Weight of 1000 grains (X3), Number of Grains (X4) and N-Fixing Bacteria (NFB)
Population (X5).

Unstandardized Standardized Collinearity

Std. t Sig. Correlations Part VIF
Coefficients Coefficients Partial Statistics
Error Zero-Order
B Betas Tolerance
(Constant) −0.184 0.186 −0.989 0.328
N Uptake 0.001 0.002 0.024 0.667 0.509 0.484 0.102 0.017 0.480 20.082
Number of 0.006 0.006 0.038 10.162 0.252 0.489 0.176 0.029 0.597 10.674
Panicles
1000-Grain 0.028 0.013 0.088 20.109 0.041 0.669 0.309 0.053 0.363 20.752
Weight
Number of 0.003 0.000 0.886 18,548 0.000 0.985 0.944 0.465 0.275 30.633
Grains
Population of 0.003 0.003 0.025 0.900 0.373 0.502 0.138 0.023 0.887 10.128
NFB

The results of the correlation coefficient and determination tests are presented in
Table 13. They reveal that the correlation coefficient (R) has a value of 98.70%. This value
indicates a strong linear relationship between the number of grains, weight of 1000 grains,
number of panicles, N uptake, and the population of N-fixing bacteria to the grain yield.
Additionally, the coefficient of determination (R2 ) is 0.974, explaining that 97.40% of the
variation in the upland grain yield can be attributed to the linear relationship with the
number of grains, 1000 grain weight, number of panicles, N uptake, and population of
N-fixing bacteria. The remaining 2.60% is influenced by other factors not analyzed in this
study. Further analysis to better understand the results of the correlation and regression
coefficients was carried out by path analysis. This analysis is helpful to assist in knowing
which variables have a more direct effect on the dependent variable. Therefore, the path
analysis was carried out to determine the direct effect of the N uptake, NFB population,
grain per panicle, 1000 grain weight, and amount of grain on grain yields (Figure 4).

Table 13. Correlation Coefficient and Determination.

Summary Models
Model R R Square Adjusted R Square std Error of the Estimate
1 0.987 a 0.974 0.971 0.18914
a Predictors: (Constant), the number of grains, the number of panicles, N-fixing bacteria, N uptake, Weight of
1000 grains.

The results of the path analysis showed the direct effect of the N uptake, NFB pop-
ulation, 1000-grain weight, number of panicles and number of grains on the grain yields
(Figure 4). The practical contribution of the independent variable to the dependent variable
was obtained by multiplying the value of the beta coefficient by the zero-order value. The
practical contribution of the N uptake and NFB population was 2%, the 1000-grain weight
was 6%, the number of panicles was 2%, and the number of grains was 88%. The results
above showed that there was an influence of the N uptake, NFB population, 1000-grain
weight, number of panicles and number of grains on the grain yields.
Agriculture 2023, 13, x FOR PEER REVIEW 18 of 22
Agriculture 2023, 13, 1987 17 of 21

Figure
Figure4.
4.Contribution
Contributionof
ofAttributing
AttributingFactors
Factorsto
toGrain
GrainYield
Yieldwith
withPath
PathCoefficient
CoefficientValue
Value(β).
(β).

4. Discussion
The results of the path analysis showed the direct effect of the N uptake, NFB
population, 1000-grain
Biofertilizers play weight,
a crucialnumberrole inof panicles and
enhancing number
bacterial of grains by
populations on providing
the grain
yields (Figure
essential 4). Thefor
nutrients practical contribution of the
soil microorganisms. Theseindependent
microbes variable to the dependent
require nutrients such as
carbon, was
variable nitrogen,
obtainedorganic ions, and water
by multiplying to thrive.
the value of the Combining
beta coefficient the biofertilizers
by the zero-order with
inorganic
value. Thefertilizers
practicalcreates an appropriate
contribution of the N composition
uptake and thatNFB
fulfills the nutritional
population was 2%,needs of
the
microorganisms
1000-grain weight[47–49].
was 6%,The the provision
number ofofpanicles a combination
was 2%,of a biofertilizer
and the number and inorganic
of grains was
fertilizers
88%. The is the right
results abovecomposition
showed that to meet theremicrobial
was annutrition.
influenceIncreasing
of the N the number
uptake, NFB of
nonsymbiotic
population, nitrogen-fixing
1000-grain weight,bacteria
numberinof thepanicles
soil is expected
and number to supply moreon
of grains nitrogen
the grainfor
plants. A higher population of N-fixing bacteria indicates elevated biochemical activity
yields.
and an improved soil quality. Consistent with other findings, research has reported that
biofertilizer
4. Discussiontreatments have a positive impact on the population of microorganisms in the
rhizosphere, leading
Biofertilizers play to an increase
a crucial role inin enhancing
the population of N-fixing
bacterial populations bacteria [50,51]. essential
by providing
Bacteria
nutrients used
for soil as biofertilizers,
microorganisms. suchmicrobes
These as inoculant
requireBacillus, can significantly
nutrients such as carbon, enhance the
nitrogen,
soil nitrogen
organic ions, and levels
waterwithout
to thrive.direct interaction
Combining with other organisms
the biofertilizers with inorganic [52]. The introduction
fertilizers creates an
of nitrogen-fixing
appropriate bacteria
composition intofulfills
that the growing media increases
the nutritional needs ofthe likelihood of nitrogen
microorganisms [47–49]. avail-
The
ability due
provision of to their role inof
a combination aiding nutrientand
a biofertilizer provision
inorganicthrough nitrogen
fertilizers is the fixation from the air.
right composition to
The rate
meet of nitrogen
microbial fixation
nutrition. is influenced
Increasing the number byofvarious factors,nitrogen-fixing
nonsymbiotic including thebacteria soil temperature,
in the soil
isthe
expected
abilitytoofsupply
the host more nitrogen
plant for plants.
to create A higher population
a low-oxygen environment of N-fixing bacteria indicates
in the rhizosphere, the
elevated biochemical activity and an improved soil quality. Consistent
availability of photosynthate from the host plant to the bacteria, bacterial competitiveness, with other findings,
research has reported
and nitrogenase that biofertilizer
efficiency [53]. treatments have a positive impact on the population of
microorganisms in the rhizosphere,
Additionally, bacterial inoculation leads leading to antoincrease in the
an increase inpopulation
the agronomic of N-fixing bacteria
characteristics,
[50,51].
particularly influencing the root morphology, as bacteria have the ability to affect the pro-
Bacteria used as biofertilizers, such as inoculant Bacillus, can significantly enhance the soil
duction of endogenous indole-3-acetic acid (IAA) by regulating auxin responsiveness [54].
nitrogen levels without direct interaction with other organisms [52]. The introduction of
Bacteria play abacteria
nitrogen-fixing pivotalinto rolethe
in growing
promoting mediatheincreases
plant root thegrowth
likelihoodby signaling
of nitrogenthe formation
availability of
due
hormones. When bacteria are present in biofertilizers and inoculated
to their role in aiding nutrient provision through nitrogen fixation from the air. The rate of nitrogen on plants, they attach
to the epidermal
fixation is influenced cells on the root
by various surface.
factors, including Thistheattachment leads to
soil temperature, thebasal root
ability colonization
of the host plant
and
to subsequently
create a low-oxygen extends to the root
environment in thehairs, allowingthe
rhizosphere, the bacteria to
availability of synthesize
photosynthate IAA, which
from the
in turn
host plantcolonizes the plant
to the bacteria, roots.
bacterial The colonization
competitiveness, and of the basalefficiency
nitrogenase roots plays [53].a crucial role in
Additionally,
the formation bacterial
of lateral inoculation
roots [55]. leads to an increase in the agronomic characteristics,
particularly
Good influencing
agronomic the root morphology,
characteristics of riceaswill
bacteria have the ability
undoubtedly increase to affect the production
the yield components. of
endogenous
This is evidencedindole-3-acetic
by a high acid (IAA) by
number of regulating
tillers, whichauxinwillresponsiveness
also result in[54]. Bacteria
a large numberplay of
a
pivotal
panicles.roleNotin promoting
all tillers in the plant
rice are root
ablegrowth
to produceby signaling
panicles, thebut
formation
it is hoped of hormones.
that with When
many
bacteria are present in biofertilizers and inoculated on plants, they attach to the epidermal cells on
tillers the number of panicles will also be large. In line with this opinion, the c2 n1 (soil
the root surface. This −attachment leads to basal root colonization and subsequently extends to the
application 10 L ha 1 + 50 kg ha−1 ) treatment produced the highest number of tillers and
root hairs, allowing the bacteria to synthesize IAA, which in turn colonizes the plant roots. The
could also produce the highest number of panicles. The results are the same as the research
colonization of the basal roots plays a crucial role in the formation of lateral roots [55].
by [56]
Goodwith the addition
agronomic of biological
characteristics of ricefertilizers using Bacillus
will undoubtedly increasepumilus
the yieldisolates which were
components. This
also able to increase the number of panicles and milled dry grain
is evidenced by a high number of tillers, which will also result in a large number of panicles. compared to the treatment
Not all
Agriculture 2023, 13, 1987 18 of 21

without biological fertilizers. Nitrogen plays an important role as a protein constituent

which will then be used by plants to increase the number of panicles per clump [57]. The
results of another study state that when plants begin to flower, most of the photosynthetic
results are distributed to the generative parts of the plant (panicles) in the form of flour [58].
Furthermore, there is the mobilization of carbohydrates, proteins, and minerals in the
leaves, stems, and roots that contribute to the formation of panicles and ultimately, this will
affect the weight of 1000 grains and grain yield.
Observations on the weight of 1000 grains indicate that the application of the biofer-
tilizer significantly influences the increase in the weight of 1000 grains in rice. Different
doses of the biofertilizer showed significant improvements in the weight of 1000 grains
compared to the control. The increase in the weight of 1000 grains in the rice can be
attributed to the beneficial effects of the biofertilizer, which enhances the root’s ability to
absorb nutrients and ultimately improves the harvest yield. This enhancement is due to the
increased development of the root system, which plays a crucial role in nutrient absorption
and transfer to the reproductive part of the plant canopy, thereby positively affecting the
yield parameters [35]. Consistent with similar studies, the findings demonstrate that the
addition of the biofertilizer, along with a reduction of recommended inorganic fertilizer
dosage by up to 50%, can lead to a significant increase in the weight of 1000 grains by up to
11.12% [12]. Moreover, the application of nitrogen-fixing biofertilizer has shown the ability
to increase the weight of 1000 grains by 7% compared to the control [52]. These results
highlight the potential of biofertilizers in enhancing the rice grain weight, contributing to
improved crop productivity and sustainability in agriculture.
The yield of the rice plants is representative of the vegetative growth as indicated by
the weight of the plant biomass, and both the roots and stems. The presence of Bacillus sp.
as a biological agent in the vegetative growth phase is thought to help optimize rice yields.
In this study, the combination of soil application of 10 L ha−1 of biofertilizer and 50 kg.ha−1
of N dose resulted in a grain yield of 29.81 g.pot−1 . This yield was not significantly
different from the treatment of soil application of 10 L ha−1 with 100 kg.ha of N dose. This
indicates that the use of biofertilizers can reduce the use of inorganic fertilizers. In line
with other research [55], the results showed that the PGPR biological fertilizer with 50%
of the recommended dosage of inorganic fertilizer was able to produce a rice growth that
was balanced with yields at 100% of the recommended fertilizer treatment. The nutritional
content, harvested dry weight, and grain yield are parameters that have the same value as
the yield of 100% of the recommended dose of inorganic fertilizer. This effect proves the
consistency of the combined application of biofertilizer and reduced doses of inorganic
fertilizers toward increasing the rice production.

5. Conclusions
Three capable isolates were identified from dryland in Lombok Island as being N-
fixing PGPR (plant growth-promoting rhizobacteria), namely Delftia tsuruhatensis strain D9
from the field ecosystem, Delftia sp. strain MS2As2 from the rainfed areas, and Bacillus sp.
from the mixed crops. The application of the liquid biofertilizer through soil application at
a rate of 10L ha−1 + 50 kg ha−1 N resulted in the same grain yield as the treatment with
a nitrogen dosage of 150 kg ha−1 , which amounted to 29.81 g pot−1 (3.98 ton ha−1 ). This
indicates a significant increase in the grain yield of 95%, with a relative agronomic efficiency
(RAE) value of up to 235.08%. The increase in the grain yield is positively correlated with
the number of grains per plant, the number of panicles, the weight of 1000 grains, the
population of N-fixing bacteria and the N uptake. The combined factors contribute to an
R-squared (R2 ) value of 97.40%. The effective contributions to the increase in the grain
yield are determined to be 87.72% from the number of grains, 5.89% from the weight of
1000 grains, 1.85% from number of panicles, 1.2% from the population of N-fixing bacteria,
and 1.16% from N uptake.
Agriculture 2023, 13, 1987 19 of 21

Author Contributions: Conceptualization, T.S.; methodology, T.S.; software, R.T.H.; validation,

M.R.S., D.H. and I.R.A.; formal analysis, D.H.; investigation, R.T.H.; resources, T.S.; data curation,
D.H.; writing—original draft preparation, R.T.H.; writing—review and editing, I.R.A.; visualization,
I.R.A.; supervision, T.S.; project administration, M.R.S.; funding acquisition, M.R.S. All authors have
read and agreed to the published version of the manuscript.
Funding: This study was supported by Universitas Padjadjaran through the Academic Leadership
Grant program, number: 2203/UN6.3.1/PT.00/2022 (Fiscal Year of 2021/2022).
Institutional Review Board Statement: Not applicable.
Data Availability Statement: Data sharing not applicable.
Conflicts of Interest: The authors declare no conflict of interest.

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