Plant Soil (2021) 460:347–357

https://doi.org/10.1007/s11104-020-04812-w

REGULAR ARTICLE

Efficiency of phosphate-solubilizing bacteria to address

phosphorus fixation in Takhli soil series: a case
of sugarcane cultivation, Thailand
Sirinapa Chungopast & Chaisit Thongjoo &
A. K. M. Mominul Islam & Sabina Yeasmin

Received: 30 June 2020 / Accepted: 16 December 2020 / Published online: 12 January 2021
# The Author(s), under exclusive licence to Springer Nature Switzerland AG part of Springer Nature 2021

Abstract Furthermore, the PSB had the ability to promote the

Aim The efficiency test of phosphate-solubilizing bac- yield and yield components of sugarcane cultivation in
teria (PSB) biofertilizer had two objectives: (i) to study pots.
the effect of the use of PSB on phosphate solubilization
of the Takhli (Tk) soil series in soil columns and, (ii) to Keywords Takhli soil series . Phosphate-solubilizing
study their impact on sugarcane (cv. Lampang) cultiva- bacteria . Phosphorus fixation . And sugarcane
tion in pots.
Methods PSB were identified and examined for Abbreviations
phosphate-solubilizing activity on culture medium. In PSB Phosphate-solubilizing bacteria
soil columns, the available phosphorus was analyzed PVC Polyvinylchloride
from leached water. In pots, the yield and yield compo- Tk Takhli soil series
nents of sugarcane were measured at age 12 months. EC Electrical conductivity
Results The PSB belonged to the Bacillus group. The pH Potential of hydrogen
use of PSB at a rate of 64 g kg−1 in the Tk soil series Dw Dry weight
produced the highest available phosphorus at PKV Pikovskaya agar medium
30.00 mg L−1 at 90 days under the soil column. PSB GYA Glucose yeast extract agar
with chemical fertilizers according to soil analysis im- NBRIP National Botanical Research Institute’s
proved the yield and yield components of sugarcane phosphate growth medium
with increasing rates of PSB. The maximum yield of 16S 16S ribosomal RNA
sugarcane was produced with N:P:K chemical fertilizer rRNA
(47:47:47 kg ha−1) with the PSB rate of 225 kg ha−1. PCR Polymerase chain reaction
Conclusions Application of PSB can address the prob-
FIDOA Chemical fertilizer input according to soil
lem of phosphorus fixation in the Takhli soil series.
analysis

Responsible Editor: Lucas Silva.

S. Chungopast (*) : C. Thongjoo

Faculty of Agriculture at Kamphaeng Saen, Kasetsart University,
Kamphaeng Saen campus, Nakhon Pathom 73140, Thailand
e-mail: agrsrnp@ku.ac.th
A. K. M. M. Islam : S. Yeasmin
Department of Agronomy, Bangladesh Agricultural University,
Mymensingh 2202, Bangladesh
Introduction
Phosphorus is one of the main elements needed for the
growth and development of plant and crop quality. For
soils with a phosphorus-fixation problem, phosphate-
solubilizing bacteria (PSB) are capable of converting
348
phosphorus from an unavailable form into an available
form (HPO42− or H2PO4−). There are various strains of
bacteria with this ability such as Acinetobacter,
Agrobacterium, Bacillus, Burkholderia, Pseudomonas,
Pantoea, and Sinorhizobium (Babalola and Glick 2012;
Chen and Liu 2019; David et al. 2014; Istina et al. 2015;
Zhang et al. 2019). The PSB mechanism creates organic
acids (formic, acetic, propionic, lactic, glycolic, fumaric,
and succinic acid) or inorganic acids (sulfuric, nitric,
and carbonic acid) to dissolve phosphates (Kalayu 2019;
Rashid et al. 2004).
Sugarcane is one of the important economic crops in
Thailand. According to a report, in Thailand in
2018/2019, sugarcane was cultivated on approximately
2 million hectares in 47 provinces with an annual pro-
duction of 132 million tonnes. The sugarcane price was
USD 22.4 t−1 for 10 levels of commercial cane sugar
(CCS), with the price being adjusted up and down by
USD 1.3/CCS (Office of the Cane and Sugar Board
(OSCB) 2019). The bacteria Agrobacterium
radiobacter and Bacillus megaterium and the fungus
Aspergillus awamori have been reported as efficient
phosphorus solubilizers. They showed positive influ-
ences on crop growth and increased the yield of sugar-
cane by 8–10% (Shankaraiah et al. 2000). Likewise,
Bacillus megaterium var. Phosphaticum increased sug-
arcane yield by 12.6% compared to no application, and
improved the sugar yield along with juice quality
(Sundara et al. 2002). Therefore, the use of only
biofertilizer or in combination with chemical fertilizers
is an option for farmers in sugarcane production to
achieve good yields and quality juice. Additionally, it
is an environmental-friendly approach to increase crop
yields and to positively impact soil health and sustain-
able agriculture (Billah et al. 2019).
PSB biofertilizer can also be used to solve the phos-
phorus fixation problem in soils caused by precipitation
as Ca3(PO4)2 and Mg3(PO4)2 (Alam and Ladha 2004)
and to reduce the negative effects of soil calcification
(Adnan et al. 2018). In Thailand, phosphorus fixation
occurs in alkaline soil series, especially the Takhli soil
series (Tk). Tk soils are classified as loamy-skeletal,
carbonatic, isohyperthermic Entic Haplustolls. These
soil series can be found in dry highland areas of the
central, northern, and northeastern regions of Thailand.
The Tk soil formation occurs from sediment deposited
on marl or layers and shows potential of hydrogen (pH)
of a base or alkaline reaction with a neutral to moder-
ately alkaline (pH 7.0–8.0) topsoil (0–50 cm) and a
Plant Soil (2021) 460:347–357
moderately alkaline (pH 8.0) subsoil (50–120 cm), with
good drainage, and moderate to high fertility status
(Land Development Department 2014).
Currently, PSB have been used to produce commer-
cial biofertilizer for agricultural crop production. PSB
have been used for the cultivation of various plants,
including mungbean, cassava, maize, cabbage, Camel-
lia oleifera, and sugarcane (Ahmad et al. 2019; Otaiku
et al. 2019; Rafique et al. 2017; Rosa et al. 2020; Wang
et al. 2017; Wu et al. 2019). The current study used a
commercial PSB product supported by the JPP Agro
(Thailand) Company. Phosphate solubilization was eas-
ily investigated using our creative model (soil column).
We focused on Bacillus spp. as PSB for calcareous soil
and the combination of PSB with chemical fertilizer.
However, there is a lack of research on the use of PSB in
sugarcane cultivation and in the Tk soil series in Thai-
land. The aim of this study was to apply the PSB to
solve the phosphorus fixation problem of the Tk soil
series and also to test the effect of the use of PSB in
combination with chemical fertilizer on the yield and
yield components of sugarcane cv. Lampang.
Materials and methods
Soil sampling and analysis
The soil samples were collected from the Tk soil series
at a depth of 0–30 cm in Tak Fa district, Nakhon Sawan
province (coordinates 15.275192°N, 100.474549°E).
All samples were air dried on open, shady ground and
passed through a 2 mm sieve. The soil properties mea-
sured were: the pH and electrical conductivity (EC) of
soil sample by taking 1:5 ratio using pH and EC meters,
respectively; organic matter using the wet oxidation
method (Walkley and Black 1934); total phosphorus
using HClO4 digestion and the vanadomolybdate meth-
od (Department of Agriculture 2010); available phos-
phorus using the ascorbic acid method (Bray and Kurtz
1945); exchangeable potassium, calcium, and magne-
sium using atomic absorption spectrophotometry (Pratt
1965); and soil texture using the pipette method
(Department of Soil Science 2011).
PSB biofertilizer identification
A PSB commercial product was provided by the JPP
Agro Company (Thailand). PSB (10 g) was diluted into
Plant Soil (2021) 460:347–357
ranges of 10−1 to 10−7. For bacterial culture, 0.1 ml soil
solution with the appropriate concentration of PSB was
transferred onto Pikovskaya’s medium (PKV) at pH 7.0
(Pikovskaya 1948) and incubated at 30 °C for 3 days.
The amount of bacteria was counted and a single colony
was separated for DNA extraction. Then the isolates
were identified based on 16S ribosomal RNA (16S
rRNA). A NucleoSpin tissue extraction kit (Macherey-
Nagel, Germany) was used for DNA extraction. The
extracted DNA was amplified using PCR with the uni-
versal primers 27F: 5’-AGAGTTTG
ATCMTGGCTCAG-3′ and 1492R: 5’-GGTTACCT
TGTTACGACTT-3′ (Frank et al. 2008). The PCR
products were checked for purity using 1% agarose gel
and were sequenced by Macrogen Inc. (Korea) using the
primers 785F: 5’-GGATTAGATACCTGGTA-3′ and
907R: 5’-CCGTCAATTCMTTTRAGTTT-3′. The
gene sequences were aligned in the NCBI database
using the BLAST homology program to identify the
isolates. Phylogenetic analysis was undertaken using
the MEGA X software and the Kimura 2-parameter
distance model and neighbor-joining method (1000
bootstrap replicates).
Investigation of phosphate-solubilizing activity
Phosphate-solubilizing activity was studied after the
bacteria had been purified and identified from the PSB
product. The bacteria were cultured on double-layered
glucose yeast agar (GYA) consisting of glucose (10 g),
yeast extract (2 g), and agar (15 g) at pH 7.0. After
autoclaving, 10% solutions of K2HPO4 and CaCl2 were
added into the medium at 30 °C. The width of the clear
zone was measured on the 3rd and 7th day. The level of
phosphate-solubilizing activity was evaluated by the
width of the clear zone as: level 1 (0 mm), level 2 (0–
3 mm), level 3 (3–6 mm), level 4 (6–9 mm), and level 5
(> 9 mm) (Katznelson and Bose 1959).
Preparation of soil column and estimation of phosphate
solubility
A completely randomized design (CRD) was conducted
using 7 treatments with 3 replications for estimating
phosphate solubility. The treatments were: T1, control;
and T2–7, PSB rates of 2, 4, 8, 16, 32, and 64 g kg−1,
respectively. A soil column was prepared using a wood-
en base (Fig. 1a) and a PVC cylinder with a diameter of
8 cm and a height of 18 cm (Fig. 1b). The end of the
349
column was cone shaped with a diameter of 2 cm and
sealed using a cotton ball and a thin white cloth (Fig.
1c). A soil sample in the Tk soil series was passed
through a 0.5 mm sieve and 1 kg sieved soil was placed
in the column with the PSB in pellet form at different
rates according to the treatment (Fig. 1d). Water
(500 ml) was poured into each column every 10 days
for 90 days and the leachate was collected in beakers
(Fig. 1e). Available phosphorus analysis of the leachate
was undertaken using the modified Bray II method
(Bray and Kurtz 1945).
Investigation of effects of PSB biofertilizer on yield
and yield components of sugarcane
The effects of the PSB biofertilizer on the yield and
yield components of sugarcane (cv. Lampang) were
based on a CRD experiment in pots. The experiment
consisted of 9 treatments with 3 replicates as: T1, no
fertilizer applied (control); T2, chemical fertilizer ap-
plied according to soil analysis (FIDOA); T3, chemical
fertilizer according to soil analysis with PSB at a rate of
75 kg ha−1 applied (FIDOA + PSB75); T4, chemical fer-
tilizer according to soil analysis with the PSB at a rate of
150 kg ha−1 applied (FIDOA + PSB150); T5, chemical
fertilizer according to soil analysis with the PSB at a
rate of 225 kg ha−1 applied (FIDOA + PSB225); T6, 125%
of chemical fertilizer according to soil analysis applied
(FI125%-DOA); T7, 125% of chemical fertilizer according
to soil analysis with PSB at a rate of 75 kg ha−1 applied
(FI125%-DOA + PSB75); T8, 125% of chemical fertilizer
according to soil analysis with PSB at a rate of
150 kg ha−1 applied (FI125%-DOA + PSB150); and T9,
125% of chemical fertilizer according to soil analysis
with PSB at a rate of 225 kg ha−1 applied (FI125%-DOA +
PSB225).
The chemical fertilizer and PSB in pellet form were
applied according to the treatment in the 2nd and 4th
months. Sugarcane was grown in 45.72-cm-diameter
width plastic pots, with a branch of sugarcane with 3
buds per pot and watering every 15 days until the
sugarcane was aged 8 months. The sugarcane was har-
vested after one year. Data collection consisted of the
yield and yield components (stem length, stem diameter,
weight per stem, internode number per stem, fresh sug-
arcane yield, CCS (Jeenyew et al. 2013), and sugar
(Hunsigi 1993). Duncan’s multiple range test (DMRT)
was applied to assess significant differences among
treatments at P < 0.05.
350 Plant Soil (2021) 460:347–357

Fig. 1 Soil column of leaching

equipment consisting of wooden
base for laying polyvinylchloride
(PVC) cylinder (a); the PVC cyl-
inder with diameter 8 cm and
height 18 cm (b); cone-shaped
with a diameter of 2 cm covering
with cotton (c); soil samples
(1 kg) with phosphate-
solubilizing bacteria (PSB) at dif-
ferent rates (d); and the beakers
for holding leached water (e)

Results 1265 mg kg −1 exchangeable calcium (high), and

71.95 mg kg−1 exchangeable magnesium (moderate).
Properties of soil samples

Soil properties of the samples are presented in Table 1. Identified PSB biofertilizer and phosphate-solubilizing
The samples of Tk soil series were clay textured, neutral activity
in reaction (pH 7.22) and non-saline (EC 0.33 dS/m)
with a moderate level of organic matter (2.35%). Soil The characteristics of the PSB product were inves-
nutrients consisted of 102.75 mg kg−1 total phosphorus, tigated baes on counting and classifying bacterial
7.31 mg kg−1 available phosphorus (low), species. The PSB had an average number of bac-
243.94 mg kg−1 exchangeable potassium (very high), teria of 2.90 × 108 CFU gDw−1 on the PKV medi-
um. Two isolates of the PSB product (B1 and B2)
were purified and sequenced based on the 16S
rRNA gene. The gene sequence was compared
Table 1 General properties of Takhli soil series at depth 0–30 cm with the available data in GenBank using a
BLAST homology search to identify the isolates.
Property Takhli soil series
Phylogenetic analyses were also performed using
Analytical value Meaning the MEGA X software. B1 was most closely re-
lated to B. mojavensis and B2 was similar to
pH (1:1) 7.22 Neutral B. megaterium (Fig. 2). These sequences were
ECe (dS m−1) 0.33 Not salty submitted to the NCBI database and the accession
Organic matter (%) 2.35 Moderate numbers were LC588998 and LC588999,
−1
Total P (mg kg ) 102.75 – respectively.
Available P (mg kg−1) 7.31 Rather low Activity analysis of the two PSB showed different
Exchangeable K (mg kg−1) 243.94 Very high levels of clear zone in the GYA double-layered agar
Exchangeable Ca (mg kg−1) 1265 High (Fig. 3). On the 3rd day, the average width of the clear
Exchangeable Mg (mg kg−1) 71.95 Moderate zone for B. mojavensis was 2 mm which indicated the
Sand (%) 32.44 – efficiency of phosphate dissolution was at level 2. On
Silt (%) 22.69 – the 7th day, the width of the clear zone was 2.8 mm,
Clay (%) 44.87 – indicating the efficiency was at level 3. For
Texture Clay – B. megaterium, the widest clear zones were on the 3rd
and 7th days with 5.6 (level 4) and 10.5 mm (level 5),
Plant Soil (2021) 460:347–357
Fig. 2 Unknown bacteria B1 and B2 were identified belonging to
B. mojavensis (a) and B. megaterium (b) by the 16S rRNA gene.
Phylogenetic tree analysis used the MEGA X software with the
respectively. Hence, B. megaterium had higher efficien-
cy of phosphate-solubilization than B. mojavensis.
Estimated phosphate solubility in soil column
The phosphate solubility was tested in the soil column.
The application of PSB at rates of 0–64 g kg−1 in the Tk
soil series continuously increased the available phos-
phorus up to 90 days. The maximum available phos-
phorus was 30.00 mg L−1 at 90 days following inocula-
tion with 64 g of PSB (Fig. 4). The lowest available
phosphorus was in the control (no PSB).
351
Kimura 2-parameter distance model and neighbor-joining method
(1000 bootstrap replicates)
Effects of PSB biofertilizer on yield and yield
components of sugarcane
Chemical fertilizer application based on the soil analysis
values or in combination with the PSB at different rates
produced significant differences in the yield and yield
components of sugarcane (cv. Lampang). The control
treatment produced the smallest stem length and diam-
eter of sugarcane (67.48 and 2.41 cm, respectively) as
shown in Table 2. The treatment FI125%-DOA + PSB225
produced the highest stem length and diameter of sug-
arcane (151.23 and 3.21 cm, respectively) which were
similar to the results for FI125%-DOA + PSB150. The
352
Fig. 3 Different width of clear zone on double-layered glucose
yeast extract agar for B. mojavensis and B. megaterium displaying
performance of phosphate solubility. Statistically significant dif-
ferences indicated with *P < 0.05 based on Duncan’s new multiple
range test
treatments FIDOA + PSB75 and FIDOA + PSB150 pro-
duced the maximum values for weight per stem of
sugarcane (1.84 and 1.93 kg stem−1, respectively). The
highest internode number per stem of sugarcane (21.73)
was observed in FI125%-DOA + PSB225. The yield com-
ponents of sugarcane are shown in Table 3, with the
application of chemical fertilizer combined with PSB
resulting in better performance regarding fresh sugar-
cane yield, CCS, and sugar yield than the application of
only chemical fertilizer. The treatment FI125%-DOA +
PSB225 had the highest fresh sugarcane yield (5.38 kg
pot−1), CCS (11.32%), and sugar yield (609.02 g pot−1),
whereas the control treatment had the corresponding
lowest values for these parameters.
Fig. 4 Available phosphorus
from leached water using Takhli
soil series over period of 90 days
with various phosphate-
solubilizing bacteria (PSB) ratio
as shown. Statistically significant
differences indicated with
*P < 0.05 based on Duncan’s new
multiple range test
Plant Soil (2021) 460:347–357
Discussion
The Tk soil series is classified as loamy-skeletal,
carbonatic, isohyperthermic Entic Haplustolls. General-
ly, the Tk soil series has a neutral to moderately alkaline
(pH 7.0–8.0) topsoil with a depth of 0–50 cm (Land
Development Department 2014). The soil has a dense
layer of white marl resulting in the problem of phospho-
rus fixation (Mahdi and Mouhamad 2018; Taalab et al.
2019; Wang et al. 2015). Most of the marl elements are
calcium carbonate (CaCO3) and the phosphorus fixation
is governed by the activities of Ca. According to soil
analysis, the Tk soil series contained high total phos-
phorus, but had low available phosphorus. It is possible
to use PSB to solve this problem soil through secretion
of organic acid production. Six isolates of phosphate-
solubilizing strains including B. endophyticus, B.
sphaericus and Enterobacter aerogenes were isolated
from the rhizosphere of sugarcane, and increased phos-
phorus solubilization and wheat growth (Mukhtar et al.
2017). Similarly, the effects of applying both the micro-
bial inoculants, B. megaterium and B. mucilaginous, in
calcareous soil showed increasing availability of phos-
phorus (32.1%) and fruit yield (28.5%) (Zhao et al.
2019). PSB can provide an alternative to increasing
the availability of phosphorus in a calcareous soil.
Many species of soil bacteria have been reported to
have powerful phosphorus-solubilizing ability, particu-
larly in the genera Pseudomonas, Bacillus, Rhizobium,
and Enterobacter (Baliah and Begum 2015; Kalayu
Plant Soil (2021) 460:347–357 353

Table 2 Effect of phosphate-solubilizing bacteria (PSB) and chemical fertilizer on yield and yield components of sugarcane grown in
Takhli soil series at 12 months after planting

Treatment Stem length (cm) Stem diameter (cm) Weight per stem (kg stem−1) Internode number per stem

T1 = control 54.28f 2.41e 0.63f 12.15d

e d b
T2 = FIDOA 121.42 2.65 1.63 16.35c
T3 = FIDOA + PSB75 128.55d 2.78c 1.84a 16.76c
cd bc a
T4 = FIDOA + PSB150 131.58 2.83 1.93 16.85c
b a d
T5 = FIDOA + PSB225 140.67 3.11 1.32 19.53b
c b e
T6 = FI125%-DOA 134.58 2.91 1.21 17.39c
b a d
T7 = FI125%-DOA + PSB75 142.58 3.12 1.36 19.83ab
T8 = FI125%-DOA + PSB150 147.60a 3.16a 1.42cd 20.25ab
a a c
T9 = FI125%-DOA + PSB225 151.23 3.21 1.47 21.73a
F-test ** ** ** **
CV (%) 12.96 12.45 13.97 11.53

FIDOA = Fertilizer input according to soil analysis for sugarcane 75, 37.5, and 75 kg N, P2O5, and K2O per ha, respectively (Department of
Agriculture 2005), Means within the same column followed by different lowercase superscripts are significantly different according to
DMRT test at *P < 0.05 and **P < 0.01

2019; Kumar et al. 2014; Mohamed et al. 2019). Spe-
cifically, the Bacillus group is often isolated and identi-
fied (Hassimi et al. 2013; Yadav et al. 2016). The cluster
analysis in the current research showed that the highest
similarity value (100%) was for B. mojavensis
(LC588998) and B. megaterium (LC588999). Mostly,
B. megaterium is identified more than any other species
of the genera Bacillus and has been isolated from dif-
ferent soils or agricultural fields (Baliah et al. 2016;
Zheng et al. 2018). Likewise B. megaterium,
B. subtilis, and other bacteria have been isolated from
calcareous rhizosphere soils presenting as a halo zone
on NBRIP plates (Liu et al. 2015). In addition, B.
mojavensis PB-35(R11) has been used as a biocontrol
agent, plant growth promoter, and has the potential to
dissolve phosphates (Prajakta et al. 2019). Co-
inoculation of Bacillus spp. (B. pumilus,
B. amyloliquefaciens, and B. mojavensis) and Pseudo-
monas putida encouraged the growth, yield, and nutrient
uptake of plants (He et al. 2019). Furthermore, the

Table 3 Effect of phosphate-solubilizing bacteria (PSB) and chemical fertilizer on fresh sugarcane yield, CCS and sugar yield of sugarcane
grown in Takhli soil series at 12 months after planting

Treatment Fresh sugarcane yield (kg pot−1) CCS (%) Sugar yield (g pot−1)

T1 = control 1.15g 8.21g 94.42i

f f
T2 = FIDOA 3.18 9.63 306.23h
T3 = FIDOA + PSB75 3.52e 9.76f 343.55g
e e
T4 = FIDOA + PSB150 3.63 10.25 372.08f
c cd
T5 = FIDOA + PSB225 4.15 10.73 445.30d
d de
T6 = FI125%-DOA 3.97 10.42 413.67e
c bc
T7 = FI125%-DOA + PSB75 4.28 10.86 464.81c
T8 = FI125%-DOA + PSB150 5.13b 11.15ab 571.99b
a a
T9 = FI125%-DOA + PSB225 5.38 11.32 609.02a
F-test ** ** **
CV (%) 12.31 11.84 13.45

FIDOA = Fertilizer input according to soil analysis for sugarcane 75, 37.5, and 75 kg N, P2O5 and K2O per ha respectively (Department of
Agriculture 2010), CCS = commercial cane sugar, Means within the same column followed by different lowercase superscripts are
significantly different according to DMRT test at *P < 0.05 and **P < 0.01
354
current results confirmed that bacteria isolated from the
PSB were not pathogenic bacteria such as B. anthracis
that causes anthrax disease and affects herbivorous
mammals (Martin and Friedlander 2010) or B. cereus
that produces a toxin that leads to diarrhea (McDowell
et al. 2019).
Testing of phosphate solubility on culture media can
be done by measuring the clear zone width from dis-
solving calcium phosphate. It has been reported that
PKV (Pikovskaya 1948), GYA (Katznelson and Bose
1959), and NBRIP (Nautiyal 1999) media can be used
to study phosphate solubility. GYA was used in this
research to determine the level of dissolved phosphate.
The high levels of effective bacteria indicated great
phosphate solubility. The current experiment indicated
that both species of isolated Bacillus were able to dis-
solve phosphates. However, B. megaterium had better
efficiency in dissolving phosphates than B. mojavensis.
A diverse range of phosphate-solubilizing microorgan-
isms have the ability to produce organic acids (Alori
et al. 2017; Jiang et al. 2018; Saeid et al. 2018) as well as
lactic acid and malic a cid ( B. subtilis and
B. megaterium), while B. amyloliquefaciens produced
lactic acid, itonic acid, isobutric acid, and acetic acid
(Sharma et al. 2013). The isolated B. megaterium-
MH142578, Acinetobacter lwoffii-MH142579 and -
MH142580 from Egyptian calcareous soils were effec-
tive in dissolving phosphates on PKV medium by pro-
ducing salicylic acid (Abdelaziz et al. 2019).
The initial soil analysis of the Tk soil series showed
that although the soil contained high total phosphorus
(102.75 mg kg−1), the available phosphorus was low
(7.31 mg kg −1 ) in the soil (Table 1) and only
2.73 mg L−1 was detected in the leached water through
the soil column. The control treatment (without the
PSB) showed that there was phosphate solubility activ-
ity. Normally, the natural ability of bacteria in the soil
can liberate phosphorus from unavailable structures
(Fitriyanti et al. 2017; Hajjam and Cherkaoui 2017;
Saeid 2018). Therefore, the phosphate-solubilizing ac-
tivity naturally increased when the PSB was applied.
The soil available phosphorus was reported to increase
39.7% by using biofertilizer containing solid-carrier,
phosphate-solubilizing microorganisms (Fitriatin et al.
2017). At the end of the current study period (90 days),
the increased available phosphorus was 30 mg L−1 of
leaching water thorough the soil column with added
PSB of 64 g. This indicated that the available phospho-
rus increased approximately 10 times from 2.73 mg L−1
Plant Soil (2021) 460:347–357
in the leached water through the soil column without
PSB. The increase in the biofertilizer rate correlated with
the increased available phosphorus. In general, the
available phosphorus in soil solution is in the range
0.031–3.1 mg L−1 (Syers et al. 2008). However, the
available phosphorus will increase when the PSB are
combined with the carbon source or inorganic phospho-
rus sources. Bacillus PSB12 solubilized 49.92 mg P L−1
after 7 days and 198.66 mg P L−1 after 14 days in PKV
broth containing glucose as the C-source (Mukhtar et al.
2017). Using phosphate rock or raw materials (poultry
bones, fish bones, and ash), B. megaterium with the fish
bones at 5 g L−1 produced the highest concentration of
released phosphorus (483 ± 5 mg L−1) in broth culture.
(Saeid et al. 2018).
The PSB have promoted the growth and productivity
of various plants such as sesame (Jahan et al. 2013),
mustard (Kang et al. 2014), soybean (Mahanta et al.
2014), tomato (Sharon et al. 2016), wheat (Suleman
et al. 2018), and sugarcane (Safirzadeh et al. 2019). It
was reported that the Bacillus spp. as PSB was used to
enhance the absorption of phosphorus by plants relying
on the mechanism of creating organic acids in soil low
in available phosphorus (Ahmad et al. 2019; Panhwar
et al. 2013). In addition, the PSB increased the sugar-
cane yield by 12.6% and also improved juice quality
(Sundara et al. 2002). Phosphorus plays an important
role regarding the sucrose from reducing sugars that
accumulate in the stem (Siswoyo et al. 2016; Wang
et al. 2013). Options for the sustainable management
of phosphorus in sugarcane have been shown by using
PSB (Soltangheisi et al. 2019). Mixed inoculations in-
fluenced the leaf phosphorus content of sugarcane when
inoculated with B. subtilis + P. fluorescens in a
Hapludox Rhodic soil with low available phosphorus
(Rosa et al. 2020). The treatment of FIDOA + PSB pro-
moted the yield and yield components of sugarcane
grown in the Pak Chong soil series of Thailand, that
also had a phosphorus fixation problem (Chungopast
and Thongjoo 2020). These findings were consistent
with our research which added mixed Bacillus sp. (the
PSB biofertilizer) to improve the yield and sugarcane
components harvested from low available phosphorus
soils. However, in our study, the PSB was suitable to
solve the problem of phosphate fixation in the Tk series
as well as other similar soil series. Furthermore, this of
study should be conducted in the field to determine the
suitable rate for the use of PSB so that farmers can then
use that rate to ensure the investment is economic.
Plant Soil (2021) 460:347–357
Conclusions
The PSB biofertilizer, containing B. mojavensis and
B. megaterium, was effective in solubilizing phosphate
in Tk soils. The soil had high available phosphorus with
the application of 64 g PSB in the soil column. The both
Bacillus helped to reduce the phosphorus fixation prob-
lem in the soil. Although bacteria can be washed off
with the water applied in the soil column, they survived
well with continuous phosphate solubilization ability for
a period of 90 days. The PSB rate of 225 kg ha−1 with
phosphorus fertilizer produced the maximum sugarcane
yield and yield components under pot conditions. This
biofertilizer enhanced the efficiency of phosphorus fer-
tilizer in the Tk soil for sugarcane production. These
Bacillus can be applied in the form of biofertilizer to
promote sustainable agriculture as well as to improve
world food production and environmental conditions.
Acknowledgements This research was financially supported by
an Academic Development Project between the Department of
Soil Science, Faculty of Agriculture at Kamphaeng Saen,
Kasetsart University Kamphaeng Saen Campus and the JPP Agro
(Thailand) Company.
Funding This study was funded by the Department of Soil
Science, Faculty of Agriculture at Kamphaeng Saen, Kasetsart
University Kamphaeng Saen Campus and the JPP Agro
(Thailand) Company (grant number 00126/59).
Compliance with ethical standards
Conflict of interest The authors declare that they have no con-
flict of interest.
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