Effects of Phosphate-solubilizing Bacteria on Micro-Tom and Soil of

Micro-Tom Rhizosphere

Wanying Zhang

1,2,a

, Shuying Wu

1,b

, Hanshi Guo

1,c

, Wanying Wang

1,d

, Yixin Li

1,e

, Jingnan Zhang

1,f,*

and Shuiqing Zhang

3,g,*

School of Life Sciences, Zhengzhou University, Zhengzhou, 450001, China

School of Agricultural Sciences, Zhengzhou University, Zhengzhou, 450001, China

Institute of Plant Nutrition and Environmental Resources Science, Henan Academy of Agricultural Sciences, Zhengzhou,

450002, China

National Natural Science Foundation of China, Grant/Award Number: 31800440

f,*

zhangjn@zzu.edu.cn,

g,*

zsq510@163.com

Corresponding author

Keywords: Phosphate-Solubilizing Bacteria, Growth-Promoting Effect, ,Inorganic Phosphorus.

Abstract: Phosphorus bacteria fertilizer can increase the utilization of soil phosphorus and promote plant growth. In

order to provide basic information for the composite engineering strains, we selected high-efficiency

phosphorus-solubilizing bacteria (PSB) from the rhizosphere of mature corn soil. In this study, a total of six

organic phosphorus strains were obtained by NBRIP medium. Further experiments were performed on

inorganic PSBs. We measured the dissolved phosphorus ratio (D/d values), NK2 and NK3 had the best

phosphate-solubilizing effects. The D/d values of NK2 and NK3 were 2.13 and 4.35, respectively. The

results of 16S rRNA amplification and sequencing showed that the NK2, NK3 were identified as

Acinetobacter sp., Pseudomonas sp., respectively. According to the data of shaker experiment for 7 days,

the maximum phosphate solubilizing contents of NK2 and NK3 were 183.10 mg·L

-1

and 79.87 mg·L

-1

respectively, and NK2 had genetic stability. The result of pot experiment indicated that the growth attributes

and the root indexes of the Micro-Tom, as well as the content of soil available phosphorus treated by NK2

(TCP) and NK3 (TCP) were both significantly higher than CK (P<0.05). These results imply that above two

strains could promote plant growth.

1 INTRODUCTION

Deficiency of phosphorus (P) is an important

limiting factor in agriculture production. Fertilizers

or inoculants made by P-solubilizing microbes are

applied to the soil with less available phosphorus

(Oliveira, 2009), which not only effectively avoids

the excessive application of phosphate chemical

fertilizers in agriculture, but also solves the problem

of the lack of available phosphorus in the soil

(Bojinova, 2008). So, environmentally friendly

substitutes for P fertilizers are urgently needed to

avoid adverse effects on agriculture production.

To circumvent phosphorus deficiency, the

phosphate -solubilizing bacteria (PSB) could play

an important role in supplying phosphate to plants

in a more environmentally-friendly and sustainable

manner (KHAN, 2007). A massive number of

research results have proved that the soil is the main

source of PSBs. PSBs in soil generally affect the

fertility of soils through biogeochemical cycles

(Wang, 2020). It has been reported that numerous

rhizosphere microorganisms have capability of

dissolving insoluble P (Hameeda, 2008, Henri,

2008). Due to the activity of P⁃solubilizing in

rhizosphere, PSBs supply P for plants in an

environmentally friendly and sustainable manner.

Several studies under greenhouse and field

indicated that PSBs have direct impacts on soil

conditions, nutrient availability and plant growth

(Fitriatin, 2014, Hussain, 2013). Understanding the

interaction of rhizosphere and microbial community

will assist the development of inoculants with

potentially greater consistency in performance and

survival for agroforestry ecosystems, especially

using indigenous microorganisms.

254

Zhang, W., Wu, S., Guo, H., Wang, W., Li, Y., Zhang, J. and Zhang, S.

Effects of Phosphate-solubilizing Bacteria on Micro-Tom and Soil of Micro-Tom Rhizosphere.

DOI: 10.5220/0011198500003443

In Proceedings of the 4th International Conference on Biomedical Engineering and Bioinformatics (ICBEB 2022), pages 254-259

ISBN: 978-989-758-595-1

2 MATERIALS AND METHODS

2.1 Sample Collection

Soil tightly adhering to corn roots was collected from

the national long-term monitoring station for soil

fertility. Using the five-point sampling method, three

vigorous and disease-free plants in the plot were

selected. Taken about 1 kg near-root corn soil

samples from the 15 cm of depth, and they were

collected in sterile bags and transported to the

research laboratory and stored at 4℃ until further

use.

2.2 Cultivation of PSBs

Serial dilution from 10

-5

to 10

-2

was achieved by

transferring 5.0 g of soil residue solution from each

preceding attenuation stage to the next. Extracted 0.1

mL samples from the 10

-3

, 10

-4

and 10

-5

dilutions,

and placed on NBRIP (Shekhar, 2003) in triplicate

and kept at 28℃ for 72 h. An isolate forming a clear

halo zone was selected as a PSB. Then a single

colony was pick-transferred to Luria-Bertani (LB)

for further purification.

The isolated bacteria were inoculated in NBRIP

liquid medium at 28℃ and 180 r/min for 7 days, and

their phosphate solubilizing activities were

quantitatively determined and compared. The

soluble P in the mediums was measured per day. The

amount of soluble P was determined through Mo-Sb

anti-spectrophotometry method (Sundararao, 1963).

2.3 Molecular Identification of

Bacterial Strains

The DNA was extracted using a Bacterial DNA Kit

(Biomed, Beijing, China) following the

manufacturer’s instructions. The 16S rDNA genes

were amplified by PCR using the universal primers:

27F (5-AGAGTTTGATCNTGGCTCAG-3) and

1492R (5-TACGGYTACCTTGTTACGACTT-3),

and sequenced as described. The amplification

reaction mixture contained 12.5 μL of Prime STAR

Max, 1 μL of 10 μmol primer (F and R), 1 μL of

template DNA and ddH

O to make up to 25.0 μL.

The resulting products were analyzed by

electrophoresis in 2.0% agarose gel and sent to the

Sangon Biotech Company (Zhengzhou, China) for

sequencing. The 16S rDNA sequences of strain NK2

and NK3 were analyzed initially by BLASTn to

acquire the closest reference sequences.

2.4 Growth Curve of Bacteria

In microbial transformation research and industrial

production applications, the physiological activity

and transformation activity of transformed bacteria

are necessary. To quantitatively determine the

transformation rate of bacteria, the concentration and

biomass of the bacterial solution should be

accurately determined to ensure the continuous and

efficient conversion process (Li, 2003). In this

section, through the optic density (OD) value assay

of culture with vary vaccination time, a chart

between OD600 and time was established.

Configured the NBRIP liquid medium with NaCl

concentration gradients of 0, 2.5%, 5%, 10%, 15%,

and 20%, respectively, the filling volume was 50

ml/250 ml. Each concentration was set to 3 parallel.

Inoculation 1 ml (OD600=1) PSBs and cultured at

28°C and 130 r/min for 20 h. Established a chart

between OD600 and the different salt

concentrations.

2.5 Pot Experiment for Evaluation of

PSBs Application on Micro-Tom

Growth

Collected seeds of Micro-Tom (Solanum

lycopersicum L. cv Micro-Tom) from School of life

science, Zhengzhou University, Henan, China.

Seeds were surface-sterilized by soaking in 5%

NaClO solution for 10 min and rinsing with sterile

distilled water. Then transferred them to sterile

dishes filled with double-layer wet filter paper and

incubated for 7 d at 26℃ after germination.

Seedlings of uniform size were transferred to pots

(diameter 15 cm, height 18 cm) filled with 1 kg of

soil (river sand: vermiculite=1:1).

The experiment was divided into 6 treatments:

CK, NK2, NK3, TCP, NK2 (TCP), NK3 (TCP), with

six replications each. CK was un-inoculated

controls. NK2 and NK3 were soil treated with 100ml

of NK2 and NK3 (OD

600

=1), respectively. TCP was

soil treated with 1% Ca

(PO4)

. NK2 (TCP) and

NK3 (TCP) were soil treated with TCP and 100 ml

of NK2, NK3 (OD

600

=1), respectively.

In pot experiments, the effects of PSBs on

Micro-Tom and soil of rhizosphere were studied.

Different growth parameters including plant height,

biomass in the plants, the total chlorophyll contents

(SPAD value), root indexes and soil available

phosphorus were examined at 15 d and 30 d after

inoculation. Replicates were not pooled. A 5 g (dry

weight) aliquot of the sampled soil was suspended in

50 ml of sodium bicarbonate solution (PH=8.5) by

Effects of Phosphate-solubilizing Bacteria on Micro-Tom and Soil of Micro-Tom Rhizosphere

255

shock (170 rpm) for 30 min at 25°C(Olsen, 1954).

The soluble phosphorus content in soil was

evaluated by Mo-Sb anti colorimetry.

3 RESULTS

3.1 Isolation of PSB and Phosphorus

Solubility of Two Selected Strains

Selected the PSBs showing greater solubilization

(both qualitatively and quantitatively) of insoluble P

under in vitro conditions. Each isolate was purified

in LB and working cultures were maintained at 4℃.

Isolates with a larger halo zone of solubilization in

NBRIP were selected for further studies. The

dissolved phosphorus ratio (D/d) of two strains

exceeded 2.0, and these were quantitatively assayed

for phosphate solubilization potential. A total of 6

different bacterial isolates were obtained from

different samples of corn soil (Table 1).

Table 1: Dissolved phosphorus ratio of 6 isolates isolated

from the near-root corn soil samples on NBRIP.

Strain D/mm d/mm D/d

NK2 2.45±0.07 1.15±0.07 2.13±0.19 b

NK3 2.73±0.15 0.67±0.20 4.35±1.26 a

NK22 7.60±0.42 5.40±0.28 1.41±0.15 c

K2 8.40±0.28 5.75±0.21 1.46±0.01 c

K3 5.45±0.21 4.35±0.35 1.26±0.15 d

K8 5.85±0.07 4.00±0.14 1.46±0.07 c

Note: D, dissolved phosphorus circle diameter; d, colony

diameter; D/d, dissolved phosphorus ratio equal to

diameter of hydrolysis circle divided by diameter of

colony.

Bacterial strains exhibiting

phosphate-solubilizing activity are detected by the

formation of clear halo around their colonies. We

selected two PSBs and showed these morphological

characteristics in Fig.1. The colonies were circular.

Figure 1: Phosphate-solubilizing activity on NBRIP by

species of NK2 isolates (Plate left) and NK3 isolates

(Plate right).

Two of the bacterial strains exhibited higher

phosphate-solubilizing activity, we measured the

daily phosphate-solubilizing activity of the two

strains within 7 days (Fig.2). The NBRIP liquid

cultures of NK2 isolates contained 183.10 mg/L (the

highest concentration) P solubilized from insoluble

(PO4)

as the sole source of P in the medium,

followed by NK3 isolates containing 79.87 mg/L

(the highest concentration).

Figure 2: Phosphate-solubilizing activity of two PSBs.

3.2 Identification of Bacterial Isolates

Nucleotide sequencing of the 16S rDNA gene of two

selected isolates proved 98%-99% similarity with

species present in the GenBank database (Table

2)(G, 1991). According to phylogenetic analysis, the

similarity between NK2 isolates and Pseudomonas

sp. was 98.33%, and NK3 isolates was identified as

Acinetobacte sp.

Table 2: Identification results for 16S rDNA sequencing.

Strain

Gram

−

Specific name

16SrDNA

homology/%

NK2 Gram

−

Pseudomonas

NK3 Gram

−

Acinetobacter

3.3 Growth Curve of Strains

The growth curve of NK2 isolates and NK3 isolates

was shown in Figure 3 (left). The bacteria grow

slowly within 0~2h and are in the growth delay

ICBEB 2022 - The International Conference on Biomedical Engineering and Bioinformatics

256

period; after 3h, the strain proliferates rapidly and

enters the logarithmic growth phase. The curve tends

to be flat after 18h.

Figure 3: The growth curve of two PSBs.

It can be seen from Figure 3 (right) that with the

strengthening of salt stress conditions, the growth of

NK2 and NK3 strains increased firstly and then

decreased. NK2 and NK3 can tolerate a wide range

of salt concentration.

3.4 Effects of PSB on Micro-Tom

Growth

3.4.1 Growth Attributes of Micro-Tom

Two PSBs with sufficient P production were chosen

to determine their beneficial effects on Micro-Tom

growth under greenhouse conditions. The

Micro-Tom grew better than CK when NK2 isolates

and NK3 isolates were colonized in the rhizosphere

of Micro-Tom seedlings (Fig.4). The NK2 (TCP)

and NK3 (TCP) significantly promoted plant height

by 170% and 160% at 30 d. Plant height was

measured from stem base to top. Leaves at a certain

fixed node height were marked and evaluated. The

total chlorophyll contents (SPAD value) increased

by 35% and 24%, the stem biomass by 850% and

800%, the root biomass by 480% and 440%,

compared with CK.

Figure 4: Growth Attributes of Micro-Tom.

3.4.2 Root Indexes

The effects of the two PSBs in the rhizosphere of

plant, measured as root length, root surface area, and

the number of root tips in the root of plant, are

presented in Fig.5. Application of NK2 (TCP) had

the strongest effect on root length of Micro-Tom,

with an increase by 39.73% at 30 d (P ≤ 0.05)

compared with CK. NK3(TCP) resulted in an

increase of 20.10 times at 30d for root surface area.

NK3 (TCP) resulted in an increase of 1.39 times at

30d for root tips (Fig. 6). Therefore, PSBs effectively

improved root development in Micro-Tom.

Figure 5: Root indexes of Micro-Tom.

Effects of Phosphate-solubilizing Bacteria on Micro-Tom and Soil of Micro-Tom Rhizosphere

257

3.4.3 Postharvest Soil Available Phosphorus

The available phosphorus content in soil treated with

NK2 (TCP) and NK3 (TCP) was at a high level at 30

d. NK2 (TCP) and NK3 (TCP) increased

significantly relative to CK at 15 d and 30 d.

Figure 6: Soil available phosphorus.

4 DISCUSSIONS

It is an urgent thing to establish a sustainable

agriculture industry that maintains the ecological

balance of soil systems for a long time. As the basic

element of fertilizer, soil available phosphorus plays

an important role in the ecosystem. In this study,

rhizosphere soil samples from corn plants were

screened by PSB isolation. Among the

phosphate-solubilizing isolates, two efficient PSB

strains were selected for the further studies. The

strains were identified as Pseudomonas (NK2) and

Acinetobacter (NK3) by 16S rDNA sequencing

technologies.

The data presented in this paper showed that two

isolates significantly promoted the growth of the

plant seedlings and root of Micro-Tom under the

greenhouse conditions. It may be due to the greater

absorption of nutrients, especially P element. The

results in this study were similar to those reported

studies (Datta, 1982, Asea, 1988). However, some

researchers obtained contrary conclusions by

inoculating PSBs to plants. Inoculating PSBs to the

seed of Chinese cabbage will promote its growth, but

there had no effect on the absorption of P element in

plants. In fact, the beneficial effects of PSBs in plant

growth largely depend on the environmental

conditions, type of strain, host plant and condition of

soil (Khan, 2009), which cannot be tested and

evaluated separately (Liu, 2014). In this work, it has

remained a significant challenge to obtain a

complete root from the soil, which caused to too

obvious differences in the measured values of root

surface area between NK3 (TCP) and CK. There is

an urgent need to use an effective method to analyze

and survey the rhizosphere. In any case, it is

necessary to explore the mechanism of NK2 and

NK3 attained a regulating balance, promoting the

growth of plants and the development of

rhizosphere. The application of PSBs with TCP

showed an obvious effect compared to control. It is

possible that the soil environment had changed and

caused a steady increase in nutrients. It is required to

explore the effect of these PSBs either alone or in

combination with other bio-fertilizers on growth of

Micro-Tom under field conditions. Further studies

under field conditions are needed to confirm the

present findings and recommend strains for

commercial applications.

5 CONCLUSIONS

NK2 and NK3 were screened in this experiment,

which have relatively strong Phosphate-solubilizing

activity and high salt tolerance. A pot experiment of

NK2 and NK3 strains were carried out. Some

indexes of the Micro-Tom in the growth period (15

d) and flowering period (30 d) were studied. The

result of pot experiment indicated that the growth

attributes and the root indexes of the Micro-Tom, as

well as the content of soil available phosphorus

treated by NK2 (TCP) and NK3 (TCP) were

significantly higher than CK (P<0.05). These imply

that both strains could promote plant growth.

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