Isolation and Phosphate Solubilization
of Beneficial Rhizobacteria from Cassava Soil
Thunyachanok Meekaew1†, Supajit Sraphet2*†,
Nattaya Srisawad2, Kanokporn Triwitayakorn2, Rungravee Boontung3,
Saowaree Bumrung4, Chaisri Tharasawatdipipat5, Sivapan
Choo-in5 and Bagher Javadi1
1Department of
Sciences, Faculty of Science and Technology, Suan Sunandha Rajabhat University,
Bangkok, 10300, Thailand
2Institute of
Molecular Biosciences, Mahidol University, Nakhon Pathom, 73170, Thailand
3Department of
Agriculture, Rayong Field Crops Research Center, Rayong, 21150, Thailand
4Nakhon
Ratchasima Agricultural Research and Development Center, Nakhon Ratchasima,
30340, Thailand
5Department
of Applied Sciences, Faculty of Science and Technology, Suan Sunandha Rajabhat
University, Bangkok, 10300, Thailand
*For
correspondence: supajit.sra@mahidol.ac.th
†Contributed
equally to this work and are co-first authors
Received
25 October 2022; Accepted 01 December 2022; Published 30 December 2022
Abstract
Rhizobacteria
as the root colonizing microorganisms show a significant impact on plant growth
and development. The aim of this study was to isolate rhizobacteria with the
ability to solubilize phosphate (P) from cassava (Manihot esculenta Crantz) rhizosphere. Soil samples of cassava
rhizosphere of Piroon 2 (PR2) cultivar were collected at 6 months after
planting (MAP). The population of culturable P-solubilizing bacteria (PSB)
indicated that their population (1.92152 x 1011) was higher than
nitrogen fixing (N-fixing) bacteria. The N-fixing bacterial population
presented only 12% of PSB. In this study the nine better PSB were isolated from
the cassava rhizosphere. These isolates showed the lag and log phases during
the 240 and 420 min, respectively. The generation rates of bacterial isolates
were in the range of G=15.49 min (NS23) and G=35.70 min (NS24). Isolates showed
the significant difference to solubilize P on solid and liquid NBRIP medium.
The result showed that isolate NS39 was significantly better than the other
isolates. The diversity of isolates to solubilize P was in the range of 0.1 to
0.3 mg/L P. The isolates NS39 and NS14 could be better candidates for field and
biotechnological applications. © 2022 Friends Science Publishers
Keywords: Phosphate solubilizing bacteria; Rhizosphere; Cassava;
Phosphorus
Introduction
Rhizobacteria
as the root colonizing microorganisms produce the significant impact on plants.
This interaction proved to be neutral, pathogenic and beneficial (Beneduzi et al. 2012). The importance of many
beneficial symbiotic relationships of rhizobacteria have been scientifically
documented (Bloemberg and Lugtenberg 2001; Buckley and Schmidt 2003; Birkhofer et al. 2008). The term of beneficial
rhizobacteria or plant growth promoting rhizobacteria (PGPR) has been interchangeably
used (Beneduzi et al. 2012). The
population of PGPR was estimated to be 2–5% of total bacterial population in
the rhizosphere; however, this population can vary in different plant and soil
conditions (Girvan et al. 2003;
Begoude et al. 2016).
The economic importance of beneficial rhizobacteria in
plant production is mostly related to their application in biofertilizers as
well as industrial microbiology and biotechnology. Different types of PGPR were
introduced and applied successfully in the agricultural fields (Lin et al. 2012). The type of symbiotic
relationship of bacteria and plant can be either helping in absorption of
nutrients or suppressing pathogenic microorganisms.
One particular nutrient that can get great benefit from
PGPR is phosphorus. Phosphorus, beside nitrogen and potassium, is a major plant
nutrient requirement for plant growth and development. Soils in agricultural
systems, especially in tropical areas contain large amounts of phosphate (P), a
considerable part of it related to its accumulation based on the regular
application of chemical fertilizers (Malhotra et al. 2018). However, a large part of the chemical fertilizers,
which is considered as the soluble and plant can uptake that easily, rapidly
fixed and transferred to insoluble and ultimately becomes unavailable (Du et al. 2020). The phenomenon of
precipitation of phosphorus in soil is greatly dependent on pH and soil type.
Free oxides and hydroxides of Al and Fe in acidic soils are highly involved in
precipitation of P, however in alkaline soils Ca mostly have this duty to fix P
and reduce the soil content of soluble P (Hou et al. 2020). Phosphate solubilizing bacteria (PSB) showed that it
can be a great asset to solve this problem. A key significant requirement to
apply beneficial rhizobacteria or PGPR, particularly PSB, successfully is their
ability to affect properly the positive symbiosis interaction with target plant. Therefore, the continuous identification of
indigenous rhizobacteria and determining their ability is inevitable.
Cassava (Manihot
esculenta Crantz) is the most important crop after rice, wheat and maize
from the Euphorbiaceae family. It is native perennial shrub from South American
region (Francis et al. 2013).
Cultivation of cassava is for root storage and other parts, mostly in tropical
and subtropical countries (Clifton and Keogh 2016). Cassava is propagated with
stem cutting and mostly cultured in nutrient deficient acidic or alkaline soils
(Bellotti et al. 2012). Cassava
farmers usually need help to improve it from rhizospheric pathogens and
diseases (Thaikert et al. 2015; Tappiban et al.
2018).
A considerable population of bacterial species is
associated with the plant rhizosphere with the ability to solubilize P can apply to produce commercial bio fertilizers in
agricultural fields (Oleńska et al. 2020). These bacteria are not
usually adequate and applications as biofertilizers increased their number in
the rhizosphere and help them to compete better with other bacteria. Thus, in the agricultural system, especially for green
culture, inocula of plants with species and strain of more effective
microorganisms is inevitable (Basu et al. 2021). As the root
colonization is highly strain- and soil-specific (Sraphet and Javadi 2022), the
screening of natural resources to find better and reliable strain linked to
identification and detection methods are necessary for inocula production
(Hassan et al. 2019). Scientific literatures showed the effectiveness of
PSB as organic acid producer in combating pathogens (Ricke 2003). The aim of
this research was to isolate and identify rhizobacteria from cassava
rhizosphere to help in combat the rhizospheric pathogens as well as assimile and
solubilize soil nutrients. Specific objective was to find isolates with P
solubilizing ability from the rhizosphere of elite cultivar “Piroon2” that
shows high yield and low cyanide content cultivated in Thailand.
Materials and Methods
Soil sampling
Soil samples
were collected from cassava field at Nakhon Ratchasima Agricultural Research
and Development Center, Department of Agriculture, Nakhon Ratchasima (14°52'
46.9704'' N: 101°38' 56.7924'' E). The samples were taken randomly from cassava
field of Piroon 2 (PR2) cultivar at 6 months after planting (MAP) from a depth
of 15–20 cm. Three replications of soil samples were collected from the
rhizosphere part of cassava root with no non-rhizosphere soil.
Population of culturable bacteria
Soil samples
(10 g) were placed in a 200 mL Erlenmeyer flask containing 90 mL DDH2O.
The sample was shaken for 1 h and used for serial dilution and then inoculation
of petri dishes containing National Botanical Research Institute's phosphate
growth medium (NBRIP) with 1.5% agar media (Javadi Nobandegani et al. 2015). NBRIP contained L-1:
glucose, 10 g: Ca3(PO4)2, 5 g; MgCl2W6H2O,
5 g; MgSO4W7H2O, 0.25 g; KCl, 0.2 g and (NH4)2SO4,
0.1 g. The same procedure was applied for the N-free MJV medium. MJV medium
contained L-1: Mannitol, 1.0; K2HPO4, 0.4; KH2PO4,
0.4; MgSO4 7H2O, 0.2; CaCl2, 0.02; Na2MoO4,
0.002; FeCl3, 0.01; pH 7.0. Measurement of bacterial population was
based on the formula:
Population of bacterial soil = bacterial colony (colony
forming unit) × dilution factor
Isolation of beneficial bacteria
Bacterial
isolates were obtained from soil samples surrounding the cassava rhizosphere. A
10 g of soil was diluted in 95 mL of sterile water to form a serial dilution up
to 10-6. Then, 0.1 mL of the final three dilutions was individually
plated on NBRIP media. Bacteria represent clearing halo-zone on the plates were
selected and purified on the same media. Nine bacterial isolates were selected
for further study based on the high solubilization of P. Colonies of bacterial
isolates were observed under the microscope. The colonies on the medium were
observed and purified during incubation for 7 days (Javadi Nobandegani et al. 2014).
Gram staining
The
conventional Gram staining method was applied for staining the colonies of
isolates. Briefly, one drop of bacterial culture was put on a slide. In order
to fix the bacteria to the slide, the slide was passed through the flame of a
Bunsen burner. The primary stain (crystal violet) and secondary stain
(safranin) as well as iodine (mordant) added in a timely manner to
differentiate the isolates and stain them. Finally, the slides were observed
using a compound microscope at 10, 40, 100 to 1000x magnification (Harrigan and
McCance 2014).
Bacterial growth curve
An inoculum
from bacterial culture was streaked on agar plate by using a sterile loop and
incubated at 37°C for 24 h. Single colony of each strain was selected,
inoculated into a test tube containing 10 mL of LB broth and incubated at 37°C
overnight. Then, 5 mL of bacterial culture were transferred into 100 mL of LB
broth in a sterile 250 mL conical flask and incubated at 37°C. The optical
density (OD) of culture suspension was measured at 0 h and every 60 min at a
Fig. 1: The pai chart represented the phosphate
solubilizing bacterial population in orange and Nitrogen fixing bacterial
population in blue
wavelength of
600 nm using a spectrophotometer. The plot of time in min on X axis versus
optical density at 600 nm on Y axis was drawn to obtain a growth curve of
bacteria. The bacterial generation rate was calculated with equation:
Determination of P-solubilization on
plates and in liquid culture
The isolates
were cultured in liquid of NBRIP medium overnight (OD600=1) followed by culture
in the wet autoclave disk of overnight re-cultured on plate of NBRIP (1.5%
agar/L). The cultured plates of NBRIP were incubated at 37ºC. The halo-zone was
measured at 48 h. For determination of phosphorus in liquid the isolates were
cultured in flask containing 100 mL NBRIP and incubated in shaker incubator at
37oC, 1500 rpm, the measurement was done after 7 days followed the
Fiske and Subbarow (1925) method with some modification.
Experiment design and statistical
analysis
The bacterial
growth curve and determination of phosphate experiments were done in a
completely randomized design (CRD) with three replicates. Data were analyzed
for variance (ANOVA) and Tukey ‘s multiple comparison with SPSS software
version 21 (IBM 2021).
Results
Soil sampling and population of
culturable bacteria
The soils
sampling was done randomly based on the standard procedures and results
indicated that PSB population was higher than N-fixing bacteria. The population
of PSB in one-gram soil was around 1.92152 × 1011 that was 88% more
than the N-fixing bacteria. The population of N-fixing bacteria in 1 g soil was
25 × 108. N-fixing bacteria represented 12% of the PSB population
(Fig. 1). It is mentioned here that some bacteria might be in both populations.
Moreover, these data presented the only culturable bacteria, which mostly
represent the whole bacterial population.
Isolation of beneficial and
P-solubilizing bacteria
A total of
nine better isolates were purified during this stage. It should mention here
that the sensitivity of the nitrogen fixing bacteria was more than
P-solubilizing bacteria. As the growth conditions of PSB were not disturbed
they were more resistant to harsh conditions. In the purification stage of PSB
they showed great solubilization of P on plate (Fig. 2). Isolates NS12, NS14,
NS22, NS23, NS24, NS26, NS29, NS32 and NS39 that showed higher activity on
NBRIP media (after purification by re-culturing) were chosen for further
experiments.
Gram staining
Nine bacterial
isolates purified on the solid NBRIP medium, were identified with Gram-staining
(Fig. 3). After reviewed under the microscope all of the bacterial isolates
showed Gram negative bacilli shape. Gram staining involved in staining the
bacterium cell wall.
Bacterial growth
Bacterial
growth curves were prepared to find the lag, log (exponential) and stationary
phases. The bacterial isolates showed the lag and log phases during the 240 and
420 min, respectively (Fig. 4). The specific time for entering these critical
phases was different among the isolates. The rate of growth of bacteria was
different, which ranged from G=15.49 (NS23) to G=35.70 min (NS24). After 420
min the bacteria started to enter the stationary phase. Furthermore, the
comparison of the bacteria growth showed that there was non- significant difference between the bacteria growth curve (P≤ 0.05) (Fig. 4).
Fig. 2: Bacteria on
NBRIP medium during the early stage of purification and re-culture
Fig. 3: The gram
staining of isolates
Fig. 4: Bacterial growth curves
Determination of P-solubilization on
plate (halo-zone) and in liquid culture
All isolates
showed differential ability of P solubility and halo-zone formation on NBRIP
media (1.5% agar/L) when incubation at 37ºC for 48 h. NS14 bacterial isolate
showed a higher performance and was significantly different compared to others
(P ≤ 0.05). Isolates NS39 and NS26 showed
no significant difference (Fig. 5).
Bacterial isolates NS24, NS32 did not show the clear halo-zone although
they showed growth on the plates, and were therefore selected for determination
of P solubilizing inliquid culture. In liquid
culture, the phosphate solubilization by isolates ranged from 0.1 to 0.25 mg/L
P (Fig. 5–6). Not all isolates showed this ability in liquid culture. The
result showed that isolate NS39 was significantly better than other isolates.
However, the isolate NS14 showed no significant difference from NS22. The
results of halo-zone and liquid culture were consistent for most of the
isolates. Surprisingly, the isolate NS12 showed no P solubilization in liquid
culture, however it showed halo-zone. Comparison of these experiments showed
different mechanism of P solubilization with isolated bacteria from the cassava
rhizosphere. (Fig. 5).
Discussion
Here we showed
the diversity of PGPR in Cassava soils, and with the help of identified PSBs,
better and more effective strains of PSB, isolated. The production of organic
acids is the main mechanism in bacteria to solubilize the soil’s mineral P. The
colonization of two important genes (PQQ and gabY genes) for organic acid
production was reported (Granada et al. 2018). Organic acids that are
involved and reported in P-solubilizer bacteria are succinic, isovaleric,
glycolic, lactic and oxalic as well as isobutyric, malonic and acetic acids
(Kour et al. 2020). As organic acids have inhibitory effect on PSB
growth (Hsiao and Siebert 1999; Pinhal et al. 2019), therefore isolated
PSB growth condition on three different lag, log and stationary phases were
investigated in more details. Overall, the bacterial isolates did not show any
significant difference in lag phase, however the specific rate of growth was
different and very low compared to the log phase (Fig. 4). This can be related
to the bacterial acclimatization to the broth medium, biosynthesis of enzymes
and other metabolites and finally adjust to the medium (Trivedi et al. 2010; Engelkirk et al. 2020).
The bacterial cells are metabolically active, however,
the increase in bacterial cells was in very low rates compared to the log
phase. In contrast, the bacterial isolates showed very high rate of growth in
log phase, as the population double in very fast pried of time (Fig. 4).
Generally, in this phase the bacteria are sensitive to external stress and as
long as constant nutrient and environment conditions provide, the fast growth
is obtained. The stationary phase between the bacterial isolates was observed
as soon as the essential nutrients in medium and water oxygen became
insufficient (Sastry and Bhat 2016).
Duration of each phase was different among the bacterial
isolates however the bacterial growth curve was not significantly different
(Fig. 4). The information provided here can help in inoculation of number of
bacterial isolates for enhancing the plant growth and producing the organic
acids. The information provided in this experiment would be helpful for
bacterial growth modeling and DNA extraction (Shigyo et al. 2019).
The Gram stain is a basic method to differentiate
bacteria to one of two groups (Gram-positive and Gram-negative) based on the
structure of bacterial cell walls. Bacterial cell
wall is one of the important parts of the bacterium for up taking the nutrients
for survival, while the prevailing
conditions affect this structure significantly. As calcium phosphate was the important ingredient in NBRIP (made it a
selective medium) and cannot be absorbed without solubilization, therefore this
condition might force the bacteria to change the growth behavior. At first
stage the structure of the membrane would change and follow the sporulation
stage to survive. This could be due to changes in peptidoglycan membrane
transformations in the early stage of growing on the medium. Nonetheless, the
results need to be confirmed with molecular identification with more clear
details (Tocheva et al. 2013).
The determination of solubilization of P was determined
with plate halo-zone and liquid culture, however the results mostly were
contradictory (Joe et al. 2018; Teng et al. 2019). This
contradiction showed that to determine the P-solubilization in liquid culture
could be more reliable than the halo-zone formation (Billah et al. 2019). Furthermore, the bacterial isolates showed no
significant difference in growth. This indicated that for industrial
application there is a need to calculate the generation rate. Our data showed
that generation rate could be important in case of using the isolate for
production of organic acids (Mattey 1992; Hsiao
and Siebert 1999; De Filippis et al.
2018; Pinhal et al. 2019).
Beneficial effects of inoculation with P-solubilizing
microorganisms on many plants have been documented by different researchers
(Billah et al. 2019; Rafi et al. 2019). The PSB with the ability
to fix nitrogen symbiotically are present in the legume nodules. Several
publications have showed that P-solubilizing strains of Rhizobium increased
growth and P content of many leguminous plants (Kalayu 2019). Mixed cultures or
co-inoculation microorganisms showed the most effective way to produce incula
for different plants. Therefore, good synergistic interaction between vesicular
arbuscular mycorrhizae (VAM) and PSB exists (Soumare et al. 2019). This
effect was also observed in inoculation with PSB and Azospirillum or
Azotobacter (Alaylar et al. 2020).
Previously
P-solubilizing bacteria have been isolated from rhizosphere by plating serial
dilutions of soil extracts in many media, although the first medium was
introduced as the Pikovskaya’s solid medium (Suleman et al. 2022). Most
PSB media contain insoluble tri- or bi-calcium P, allowing the isolation of
P-solubilizer microorganisms by the formation of “halo” zone around their
colonies (Fig. 5). This halo-zone is
Fig. 5: Phosphate solubilization on NBRIP
plates to measure halo-zone (orange) and NBRIP liquid culture (blue) by the
isolates. Means with different letters represent significant difference at P < 0.05 Tukey’s multiple comparison,
n = 3. Bars show standard error of the treatment’s mean
Fig. 6: Setup for bacterial isolation during
phosphors determination by developing blue color before measuring the optical
density at 882 nm
due to secretion of organic acids or protons, which drop
the medium pH. Although there is instability of P-solubilization by some
isolates, most isolates may retain their ability for long time after culturing
(Ahmad et al. 2022). The identification of better and effective P
solubilizers should be done by measuring their P-solubilizing capacities in
liquid cultures as determined in our research (Fig. 5 and 6). For better
isolation of PSB, NBRIP medium was used, which is known to show higher
efficiency compared to other PSB isolation media (Patre and Peter 2021).
Conclusion
The cassava
soils entailed a high diversity of PSB. Ability of isolated bacteria to
solubilize P (in particular calcium phosphate) in harsh conditions could direct
that they could be better candidates for biofertilizers production even for
other plants. Molecular identification and characterization of bacterial
isolates as well as field survey may help finding clearer distinction for field
applications in future.
Acknowledgments
This research
project was supported by Mahidol University, Thailand. The authors would like
to thank the Faculty of Science and Technology, Suan Sunandha Rajabhat
University (SSRU), Thailand. Special thanks to the Chemistry group (Department
of Sciences, SSRU) and Science Center (SSRU) for their generous support.
Institute of Molecular Biosciences, Mahidol University, Salaya Campus, Thailand
are gratefully acknowledged for their kind support.
Author Contributions
KT, NS, RB,
SB, CT and SC: Conceptualization, investigation and methodology; TM: Microbial
experiments; SS and BJ: Supervision, review and editing
Conflict of Interest
The authors
declare that they have no competing interests
Data Availability
Data presented
in this study will be available on a fair request to the corresponding author
Ethics Approval
This work does
not involve animals hence ethics approval not required
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