土壌リン動態とトウモロコシ苗のリン吸収におけるリン溶解細菌の役割

The role of phosphate solubilizing bacteria in soil phosphorus dynamics andphosphorus accumulation of maize seedlings

学

生

氏

名

： ：龍

学

生

番

号

： ：

寒
Ｄ203523

主 指 導 教 員 ： ：和 崎淳

副 指 導 教 員 ： ：中 坪

孝 之

副 指 導 教 員 ： ：上 田

晃 弘

—— Contents ——Abstract .................................................................................................................................................................... i
Chapter I Introduction .............................................................................................................................................1
1.1 Background ...............................................................................................................................................1
1.1.1 Phosphorus (P) ...............................................................................................................................2
1.1.1.1 What is P .............................................................................................................................2
1.1.1.2 P in soils ..............................................................................................................................4
1.1.1.3 Supply of Soil P - Phosphate Fertilizer .............................................................................. 9
1.1.2 Phosphate solubilizing bacteria (PSB) ........................................................................................ 12
1.1.2.1 What is PSB ......................................................................................................................121.1.2.2 Mechanism of PSB release P ........................................................................................... 15
1.1.2.3 What can PSB do for agriculture ......................................................................................18
1.2 Research purpose ......................................................................................................................................0
1.3 Research frame ......................................................................................................................................... 1
Chapter II Isolation and screening of PSB ............................................................................................................. 4
2.1 Materials and methods ..............................................................................................................................4
2.1.1 PSB separation and identification ................................................................................................. 4
2.1.1.1 PSB separation ....................................................................................................................5
2.1.1.2 PSB DNA extraction and identification ............................................................................. 7
2.1.1.3 Strain Preservation ............................................................................................................. 8
2.1.2 PSB pre screen ...............................................................................................................................92.1.2.1 Shake flask culture ............................................................................................................. 9
2.1.2.2 P concentration of culture solution measure .................................................................... 10
2.1.2.3 PSB soil viability test ....................................................................................................... 10
2.1.3 PSB release P from phosphates in shake flask ............................................................................10
2.1.3.1 Shake flask culture ........................................................................................................... 10
2.1.3.2 P concentration of culture solution measure .................................................................... 11
2.1.3.3 pH of culture solution measure ........................................................................................ 11
2.1.3.4 Organic acid of culture solution detection ....................................................................... 11
2.1.4 Data analysis ................................................................................................................................12
2.2 Results .....................................................................................................................................................122.2.1 PSB identification ........................................................................................................................122.2.2 PSB pre screen .............................................................................................................................15
2.2.3 Five PSB strains’ information ..................................................................................................... 18
2.2.4 PSB strains release P from phosphates in shake flask ................................................................ 18
2.2.4.1 Strain A release P from phosphates in shake flask ...........................................................18
2.2.4.2 Strain B release P from phosphates in shake flask ...........................................................21
2.2.4.3 Strain F release P from phosphates in shake flask ........................................................... 23
2.2.4.4 Strain G release P from phosphates in shake flask .......................................................... 26
2.2.4.5 Strain H release P from phosphates in shake flask .......................................................... 28
2.3 Discussion ...............................................................................................................................................30
Chapter III PSB release P from sterilized soils (inoculation experiment) ........................................................... 36
3.1 Materials and methods ............................................................................................................................36
3.1.1 Materials and treatment ............................................................................................................... 36

3.1.2 Soil P fractions measuring ...........................................................................................................39
3.1.3 Soil pH measuring ....................................................................................................................... 40
3.1.4 Soil organic acid detection .......................................................................................................... 40
3.1.5 Soil phosphatase measuring ........................................................................................................ 40
3.2 Results .....................................................................................................................................................43
3.2.1 Strain A release P from soils ........................................................................................................43
3.2.2 Strain B release P from soils ....................................................................................................... 50
3.2.3 Strain F release P of soils ............................................................................................................ 57
3.2.4 Strain G release P of soils ............................................................................................................63
3.2.5 Strain H release P of soils ............................................................................................................68
3.3 Discussion ...............................................................................................................................................73
Chapter IV PSB affect maize seedling P accumulation in sterilized soils (sterilized co-culture experiment) ....91
4.1 Materials and methods ............................................................................................................................91
4.1.1 Materials and treatment ............................................................................................................... 91
4.1.2 Seedling culture ........................................................................................................................... 92
4.1.3 Seedling P content measuring ..................................................................................................... 94
4.1.4 Soil P fraction, pH, organic acid and phosphatase measuring .................................................... 94
4.2 Results .....................................................................................................................................................95
4.2.1 Maize seedling P accumulation of two soils ............................................................................... 95
4.2.2 Soils pH ....................................................................................................................................... 98
4.2.3 Soils organic acid ........................................................................................................................ 99
4.2.4 Soils phosphatase ...................................................................................................................... 102
4.2.5 Soils fractions ............................................................................................................................ 105
4.3 Discussion .............................................................................................................................................109
4.3.1 PSB inoculation affect maize seedling P accumulation ............................................................109
4.3.2 Exploration of Factors Influencing Maize Seedling P Accumulation ...................................... 112
Chapter V PSB affect maize seedling P accumulation in natural soil (non-sterilized co-culture experiment) . 118
5.1 Materials and methods ..........................................................................................................................118
5.1.1 Materials and treatment ............................................................................................................. 118
5.1.2 Soil pH, organic acid and phosphatase measuring ....................................................................123
5.1.3 Seedling P content measuring ................................................................................................... 123
5.1.4 Soil DNA extraction .................................................................................................................. 124
5.1.5 Amplicon sequencing ................................................................................................................ 124
5.1.6 Bioinformatic Analysis ..............................................................................................................124
5.2 Results ...................................................................................................................................................124
5.2.1 Soil physicochemical indicators ................................................................................................124
5.2.2 Maize seedling physiological indicators ................................................................................... 127
5.2.3 Rhizosphere bacteria species annotation and taxonomic ..........................................................128
5.2.4 Rhizosphere bacteria diversity .................................................................................................. 131
5.2.5 Difference of rhizosphere bacteria between groups ..................................................................133
5.2.6 Correlation network of rhizosphere bacteria .............................................................................135
5.2.7 Functional genes prediction ...................................................................................................... 136
5.2.8 Canonical Correspondence Analysis of Environmental Factors and Maize Seedling P
accumulation .......................................................................................................................................138
5.3 Discussion .............................................................................................................................................140
Chapter VI General Discussion .......................................................................................................................... 145

6.1 PSB P release ability .............................................................................................................................145
6.2 Maize seedling P accumulation ............................................................................................................ 148
6.3 Maize rhizosphere bacteria community ............................................................................................... 149
6.4 Conclusion and Perspectives ................................................................................................................ 150
References ...........................................................................................................................................................154
Acknowledgment ................................................................................................................................................166
Appendix .............................................................................................................................................................167
I. Abbreviation ............................................................................................................................................ 167
II. Culture media and solution ....................................................................................................................168
III. Equipment .............................................................................................................................................168
IV. Measuring methods ...............................................................................................................................169

Abstract
Phosphorus (P) is a macronutrient element with low availability in soil, which
restricts plant growth. Several studies have indicated that phosphate-solubilizing
bacteria (PSB) can help for converting unavailable P in the soil and promote plant
growth. However, few reports have been found on the differences in P release by
PSB strains in red soil (La) and cinnamon soil (Ci), nor have there been reports on
the differential effects of PSB on P accumulation in maize grown in these two soils.
These two soil types are highly important as arable land types in China and possess
different P fractionation and physicochemical properties. This study aims toinvestigate the role of PSB in soil P dynamics and maize seedling P accumulation,and to determine the similarities and differences in P fractionation changes caused
by PSB and P accumulation in maize seedlings in these two soils.
This study revealed that while different PSB strains might cause variations in
P fractionation, the patterns of change within the same soil remained consistent.
For La, which had a pH of 5.0-5.5 and high iron and aluminum content, the P
fractionation was predominantly influenced by moderately labile P (M labile P).
Through PSB inoculation, regardless of the provision of tri-calcium phosphate(TCP), the released P was primarily used to increase the content of M labile P, with
only a small amount entering the labile P fraction. On the other hand, for Ci with a
pH of 8.0 and high calcium phosphate content, the P released by PSB mostly
entered the labile P fraction, enhancing the soil's P availability to plants. Therefore,
it can be concluded that the effects of PSB strains on soil P release and P
fractionation were primarily controlled by soil type. Although the five strains usedi

in the experiment exhibited different abilities to release phosphates, the trends ofchange induced by all these strains remained consistent within the same soil. In
other words, in Ci, regardless of the strain, none of them caused an increase in M
labile P beyond the increase in labile P. Similarly, in La, none of the strains caused
an increase in labile P beyond the increase in M labile P, as further confirmed by
the results of Pearson correlation analysis.
PSB strains significantly enhanced maize seedling P accumulation in bothsterilized La and Ci soils. When PSB was combinedly utilized with TCP in La, it
significantly increased maize seedling P accumulation compared to PSB alone.
However, in Ci, PSB alone showed promising results in P accumulation, and the
combination with TCP did not result in higher P accumulation than PSB alone.
Based on the results of Pearson correlation analysis, PSB inoculation was directly
and positively correlated with maize seedling P accumulation in Ci without the
supply of TCP. In La, although no direct correlation was found between the two, acertain indirect positive correlation, mediated by soil indicators, was observed. In
addition, in chapter 3, when PSB strains were inoculated into sterilized soils, PSB
inoculation was positively correlated with M labile P in La and with labile P in Ci,
while in this sterilized co-culture, PSB inoculation was positively correlated withlabile P in all cases except Ci with TCP.
PSB strains did not enhance maize seedling P accumulation in natural soil
(non-sterilized La) as significantly as soluble P did. In addition, strain A did notexhibit the same promoting effect on maize seedling P accumulation in natural La
as it did in sterilized La (chapter 4). The investigation of PSB's effect on theiirhizobacterial community of maize seedlings revealed that the inoculated strains
did not become dominant species in the rhizobacterial community. Nevertheless,
PSB inoculation still altered the structure of the rhizobacterial community.
Canonical Correspondence Analysis found a positive correlation between theabundance of Pseudomonas, to which strain G belongs, and maize seedling P
accumulation, and the abundance of dominant species in all three groups (A, F, andG) was positively correlated with PSB inoculation. When data of P treatment was
excluded, Pearson correlation analysis revealed an indirect positive correlation
between PSB inoculation and maize seedling P accumulation, which was mediatedby soil acid phosphatase activity.

iii

Introduction

Chapter I

1.1 BackgroundPhosphorus (P) is a macroelement for plants and is one of the limiting factors
restricting their growth (Aerts and Chapin, 1999; Raghothama, 2005). Although it
widely exists in the soil (Larsen, 1967), only a small part can be absorbed by plants
directly. P is mainly present in unavailable forms such as insoluble and organic
forms. Insoluble P in soils can be mobilized by plant and microbial functions.
Since scientists found that some bacteria can dissolve insoluble natural raw rockphosphate in the early 20th century (Sackett et al., 1908), research on
phosphate-solubilizing bacteria (PSB) has been widely carried out in various
aspects (Khan et al., 2009; Ingle and Padole, 2017). It has been shown that a wide
range of PSB isolates contributes to transforming unavailable P in soils andsupporting plant growth. Some studies have focused on PSB themselves, such as
the strains' function and mechanism of degrading phosphates (Amy et al., 2022;
Ding et al., 2021), as well as PSB population and its dynamics (Djuuna et al.,2022). Studies involving crop productivity have also been conducted, including theapplication to transform soil unavailable P (Alam et al., 2021; Liu, X. et al., 2021)
and supply P to crops (Khan et al., 2022; Alam et al., 2022), and even to improvesoil quality (Dasila et al., 2022; Pathak et al., 2021).

1

1.1.1 Phosphorus (P)1.1.1.1 What is P
Phosphorus (P) is a geochemical element with atomic number 15, represented
by the symbol P. P ranks approximately tenth in the Earth's elemental reserves,
with a content of around 0.12% (Rudnick et al., 2003). It primarily exists in thenatural environment of the Earth in the form of phosphate minerals such as apatite
and calcite, which are commonly found in rocks (Li et al., 2019; Decrée et al.,2020; Dong et al., 2020). As rocks containing these minerals undergo weatheringand erosion processes, phosphates are slowly released into the soil and water,
making them available in other forms or more easily accessible to organisms (Sunet al., 2005). Alongside the nutrients brought about by rock weathering, thedecomposition of feces and animal and plant carcasses also serves as an importantsource of soil P nutrition (Ito et al., 2010; Kear, 1963) (Fig. 1.1.1.1).
P is one of the major chemical elements of life, forming the backbone of DNA
and RNA molecules and serving as the primary source of energy in cells. It
participates in various metabolic pathways such as phosphorylation, signaltransduction, and fatty acid metabolism. Plants absorb P in the form of H2PO4- andHPO42- and utilize it for synthesizing nucleic acids, phospholipids, and coenzymes.
They regulate the activity of diverse enzymes through phosphorylation and use P
to synthesize phosphoric esters with sucrose, facilitating the transport of sucrose
within plants. Insufficient P in plants leads to metabolic blockages, resulting instunted growth, reduced tillering, delayed maturity, and decreased reproductive
ability. The presence of a purple-red coloration in the stems or leaves often2

indicates P deficiency (Pan, 2004; Marschner, 2011).
A study conducted by Mollier et al. (1999) found that when maize plants are
deficient in P, leaf area expansion and root development are significantly hindered.

Fig. 1.1.1.1 Phosphorus cycling in soils
Although root development is slightly stimulated in the first few days of P
starvation, it ultimately cannot grow well due to the inhibition of leaf development
caused by P deficiency, leading to a decrease in photosynthesis. According to thefindings of Zhao et al. (2013), the deficiency of P is a factor that contributes to the
manifestation of symptoms in citrus huanglongbing disease, and supplying P to
diseased trees helps alleviate HLB symptoms. Lovelock (2006) discovered through3

the study of mangrove physiological processes that insufficient P inhibits normal
physiological development and photosynthesis of mangrove plants.
Plants absorb phosphorus (P) from the soil through their roots, but due tovarious factors, the amount of P absorbed by plants is limited (Bieleski, 1973;
Raghothama, 2005; Smil, 2000; Sohrt et al., 2017). In fact, plant productivity interrestrial ecosystems, including natural ecosystems and farmland, is primarily
constrained by P (Du et al., 2020; Hou et al., 2020; Vitousek et al., 2010).

1.1.1.2 P in soils
Plants mainly absorb P from the soil through their roots, and the absorbed P isin the form of water-soluble inorganic compounds (H2PO4- and a small amount of
HPO42-) that exist in the soil solution. The concentration of these P compounds is
usually around 1 ppm or lower (Mullins, 2009). However, the total amount of P in
the soil is not disappointingly low. For example, the total P content in Frigid desert
soils (Great group in Chinese soil taxonomy) in China reaches 19.1 × 10² g/m³,
Fluvi-aquic soils are 17.7 × 10² g/m³, and volcanic soils also reach 16.0 × 10²
g/m³ (Zhang et al., 2005). The common concentration of total P in global soils is
about 200-800 mg/kg of soil (Tiessen, 2008).
Soil P availability is influenced by many factors, such as soil pH, organic
matter, free iron or aluminum, calcium carbonate, and temperature (Muindi, 2019).
Once P enters the soil, it is fixed in various ways: in acidic soils, P is quickly
immobilized with iron and aluminum, while in alkaline soils, it is immobilizedwith calcium ions to form calcium phosphate or calcium magnesium phosphate
4

(Hemwall, 1957; Hsu, 1965; Bear and Toth, 1942; Cho and Caldwell, 1959).
Chang and Chu (1961) reported that in soils with a pH range of 5.3 to 7.5, most of
the P was fixed to form Al-phosphate, a portion was fixed by Fe-phosphate, andthe form of Ca-phosphate was retained the least. Mackenzie and Amer (1964)
found that in soils such as Haldimand clay, Vasey, Oneida, Honeywood, Guelph,and Minesing (pH: 5.3-7.6), the increase of Ca-P exceeded A1-P or Fe-P in
Minesing soil at pH 7.6. The potential of the soil to remove phosphate from thesoil solution is called soil Phosphorus Retention Potential (PRP) (Wild, 1950),
which is generally classified as Low, Moderate, High, and Very High. Soils with
Very High PRP are mainly concentrated in Central and South America,
Southwestern North America, Central Africa, Northern Europe, and South Asia.
Soils with Low PRP are mainly concentrated in Central North America, Central
Europe, Central Asia, West Asia, Southeast Asia, and some parts of Australia(Reich, 1998). PRP is also influenced by soil mineralogy, clay content, soil pH, aswell as climate factors such as temperature and humidity (Batjes, 2011).
In order to study the distribution of phosphorus (P) in soil more effectively
and support practical applications such as P mobilization, scientists have been
exploring increasingly rational schemes for soil P fractionation extraction anddetermination for decades.
In 1957, Chang and Jackson proposed a relatively complete soil fractionation
extraction method based on the different solubilities of various phosphate
compounds. The main steps are as follows:
1）P in the soil solution, physical adsorption P and exchangeable calcium,5obtained via 1 N NH4Cl；
2）Al-P, P combined with aluminum, obtained via leaching with 0.5 N NH4F
(forms of P extractable by this solution: Al-P completely, Fe-P slightly);3）Fe-P, P combined with iron, obtained via leaching with 0.1 N NaOH
(forms of P extractable by this solution: Al-P, Fe-P, organic-P);4）Ca-P, P combined with calcium, obtained via leaching with 0.5 N H2SO4(forms of P extractable by this solution: Ca-P completely, Al-P and Fe-P
considerably);5）O-P, reductant soluble Fe-phosphate (iron oxide occluded), occluded Al-P
and occluded Al-Fe-P.
Although this method is widely used in acidic and neutral soils, NH4F is notvery effective in distinguishing between Al-P and Fe-P. In addition, NH4F reacts
with calcite in calcareous soils to form CaF2, which strongly adsorbs P and affects
the actual measurement of Ca-P. To overcome this limitation, Chinese scientists
Jiang Baifan and Gu Yichu (1989) proposed a fractionation extraction method
specifically for calcareous soils. The main steps are as follows:
1) Ca2-P, obtained via 0.25 M sodium bicarbonate solution at pH 7.5;
2) Ca8-P, obtained via leaching with 1 M NH4Ac solution at pH 4.2;
3) Al-P, obtained via leaching with 0.5 M NH4F solution at pH 8.2;
4) Fe-P, obtained via leaching with 0.1 M NaOH-Na2CO3 solution;5) O-P (occluded-P), obtained via leaching 0.3 M sodium citrate - 0.5 g
sodium dithionite - 0.5 M sodium hydroxide solution;6) Ca10-P, obtained via leaching with 0.5 M H2SO4 solution.
67) This method subdivided calcium bound P into different types as dicalcium
phosphate (Ca2-P), octacalcium phosphate (Ca8-P) and decacalcium phosphate(Ca10-P). it is widely used in the study of P fractionation in calcareous soils in thecentral and northern regions.
Today, the soil P fractionation measuring method that was widely used is themethod proposed by Hedley in 1982. Hedley method combines the determinationof inorganic and organic P, and extracts soil P with five solutions, measuring seven
P fractions:
1) Resin exchangeable P (Resin-P) mainly contains inorganic P. This part ofinorganic P can be exchanged via anion exchange resin, plants can absorb this part
directly;2) NaHCO3 extracted P (NaHCO3-P), including inorganic P (NaHCO3-Pi)
and organic P (NaHCO3-Po). The organic P part contains the organic P dissolved in
the soil solution, which is easy to be mineralized and absorbed by plants, with higheffectiveness. Inorganic P mainly refers to the inorganic P adsorbed on the soil
surface, which is easy to be absorbed and utilized by plants, with higheffectiveness;
3) NaOH extracted P (NaOH-P), including inorganic P (NaOH-Pi) andorganic P (NaOH-Po). Sodium hydroxide extracted P mainly refers to thechemically adsorbed P on the surface of iron aluminum oxides and clay minerals,
which is very rich in soil;
4) HCl extracted P (HCl-P), including inorganic P; roughly composed ofcalcium phosphate, but also including some inorganic P which is occluded within
7

sesquioxides and released on the partial dissolution of these oxides.
5) Residual P, including inorganic P and organic P. Residual P mainly refers
to P that cannot be extracted in the previous steps and is extracted byhigh-temperature digestion, include occluded phosphates and most stable organic
phosphates.
Based on Hedley method, Yang and Post (2011) investigated the relation
between the distribution of different forms of P and the stage of soil development.
Hou et al. (2018) provide a global dataset of soil P fractions separated using the
Hedley method. Xu et al. (2018) used the Hedley method to study the nonadditiveeffects of biochar amendments on soil P composition in acidic and alkaline soil.
Hedley method was also used to analyze P fractions in Andisol and Ultisol (Delfim
et al., 2020) and plantation soil in Jiangxi, China (Liu et al., 2022).
In the Hedley method, the P extracted by resin and NaHCO3, which can bedirectly absorbed and utilized by plants, is referred to as available P. The mostwidely used direct method for measuring available P in soil is the NaHCO3
extraction method proposed by Olsen in 1954. Using the Olsen method,
researchers have reported the latest global surface soil available P content(McDowell et al., 2023). The report indicates that the content of available P in
soils is below 2 mg/kg soil in some parts of northern Africa, central and western
Asia, central Australia, and southern North America. In some areas of SouthAmerica, South Africa, southern Asia, and central Asia, it ranges from 2-5 mg/kg
soil. The proportion of land with available P greater than 40 mg is relatively low
and includes only small areas of Europe, eastern Asia, eastern North America, and8the western coast of South America. However, for cultivated land, due to humanintervention, the accumulation of P has reached a high level. Li (2011) reported
that from 1980 to 2007, the average P accumulation in arable land in China was
242 kg per hectare. Meanwhile, Olsen-P increased from 7.4 mg/kg soil in 1980 to24.7 mg/kg soil in 2007. Furthermore, 9.3% of cultivated land has exceeded a levelof 40 mg/kg soil (Li et al., 2011), which poses a risk of P leaching (Zhong et al.,2004). George also mentioned in their article that many cultivated soils worldwide
have accumulated large amounts of P that are sufficient for decades of foodproduction (George et al., 2016) but also pose significant risks to the environment
through loss and harm (Elser and Bennett, 2011). The fundamental cause of theaccumulation of P in cultivated land is the huge input of P fertilizer in agricultural
production.

1.1.1.3 Supply of Soil P - Phosphate Fertilizer
The P provided by natural soil can hardly reach the P nutritional level
required for high-density agricultural cultivation (Breman et al., 2001; Fageria andOliveira, 2014). If farming want to get a good yield in planting, the P concentrationof the soil solution should reach 1.5 μmol-P/L in wheat and rapeseed, 5 μmol-P/L
in tomato and soybean, and 7 μmol-P/L in onion (Föhse et al., 1988). In order tomake up for the shortage of P supply in natural soil, in agricultural production, P
was added to cultivated land by applying P fertilizer, but in fact, the P fertilizer
applied in agriculture is often much higher than the actual P demand of crops.
Taking vegetable cultivation in China as an example, greenhouse vegetables9harvested 44 kg-P per hectare per season, while fertilization was as high as 541
kg-P per hectare; open-air cultivated land harvests 25 kg-P per hectare per season,and the input of phosphate fertilizer reached 117 kg-P per hectare (Yan et al.,2013). In Japan in 1997, the amount of P applied to farmland soil was 6.3 times theamount absorbed by crops (Mishima et al., 2003). A small part of the P put into thesoil was used by crops, and most of it was bound to the soil and retained in the soil
(Mishima et al., 2003). The survey found that the annual accumulation of P in
agricultural land in China exceeded 39 kg per hectare (Zhang et al., 2019). A study
on the continuous application of P fertilizer in paddy soil in southern South Korea
for 31 years found that the application of chemical fertilizers did not increase theinorganic P content, but increased the organic P content, and it significantly
increased the total P of the soil (Lee et al., 2004), this research indicated thatduring 31 years of continuous cultivation, approximately 37% of P was contributed
to crop accumulation, and approximately 50% of P was accumulated in the soil.
Although the yield of agricultural products benefits from the application of P
fertilizer, the utilization rate of P fertilizer rapidly decreases uncontrollably with
the increase of P fertilizer application amount. In the past 10 years until 2014, theuse of P fertilizer in China has been continuously increasing, with an averageannual increase of 3.5 kg per hectare. However, as the use of P fertilizer increases,
the utilization rate of P fertilizer (crop P yield ÷ P input ×%) Decreased by 48
percentage points, and by 2014, the utilization rate of agricultural P fertilizer could
only remain at 20% (Zhang et al., 2019). Many studies have reported on thecritical value (The soil available P level required when the plant output reaches
1095% of the maximum output) of Olsen-P in soil. For example, the critical value for
British grasslands was 23-25 mg Olsen-P kg-1 soil (Johnston et al., 2013); The
critical value of Australian grassland was 9-15 mg Olsen-P kg-1 soil (Sandral et al.,2019); The critical value of Spanish ryegrass soil was 24 mg Olsen-P kg-1 soil
(Sánchez et al., 2015). Researches suggested that exceeded the critical value, noamount of P input could significantly increase plant output. Studies have shownthat applying P fertilizer in low P soil (Olsen-P ≤5 mg kg−1 soil) could

effectively promote an increase in absolute yield; In soils with higher P content(Olsen-P > 10 mg kg−1 soil), even applying a large amount of P cannot achieve
high P utilization efficiency (Ros et al., 2020).
In addition, numerous researchers have discovered that P, which was notabsorbed by crops or fixed in the soil, was carried away from the land throughrainwater erosion and surface runoff. The greater the application of P fertilizer, thehigher the magnitude of the loss (Cao and Zhang, 2004; Xia et al., 2008; Zhang etal., 2003). In 31 years of continuous fertilization and cultivation in South Korea,
when 37% of the P input was absorbed by plants, 13% of P might be lost throughleaching (Lee et al., 2004). In Japan, when the P utilization rate in farmland
reached 16% of the P application rate, the P loss caused by leaching from farmland
was 0.6% of the P application rate (Mishima et al., 2003). Such agricultural spills
have become a major cause of P pollution in the Earth's water system (Chowdhury
et al., 2017).
In general, P fertilizer had become one of the factors that modern agricultural
production depends on, but the utilization rate was very low. It has resulted in a11

low ratio of production input and return, raising production costs, and these highinputs have also brought about serious environmental pollution problems such as
water eutrophication (Sims and Sharpley, 2005; Sharpley et al., 2013).
In addition, the increasing inputs were depleting the Earth's P reserves, which
is becoming a serious problem as such production methods cannot support
long-term human agricultural production. Fortunately, only a small portion of the P
input in the early stages of agriculture leached out of the soil, with the majority
being retained in the cultivated soil (Lu and Tian, 2017). Whether P fertilizer input
can be combined with the development and mobilization of these retained P in thesoil to reduce the amount of P fertilizer input in the future is a question worth
exploring. In this regard, researchers have proposed a series of methods to reduce
P fertilizer application and increase P efficiency. Among them, the utilization ofphosphate solubilizing bacteria (PSB) as biological phosphate fertilizer to mobilize
soil P is an environmental and sustainable way to partially substituted chemical
phosphate fertilizer (Zhang et al., 2019; Walpola and Yoon, 2012; Dandessa andBacha, 2018; Sharma et al., 2013).

1.1.2 Phosphate solubilizing bacteria (PSB)
1.1.2.1 What is PSB
Since scientists found that some bacteria can dissolve insoluble natural raw
rock phosphate in the early 20th century (Sackett et al., 1908), research elucidating
PSB has been pursued widely (Khan et al., 2009; Ingle and Padole, 2017).
PSB can accelerate soil P cycling (Hafeez et al., 2019; Liu et al., 2022) and12

can counteract the antagonistic effects of soil calcification on bioavailable P(Adnan et al., 2017). The PSB number can be related closely to the P fraction (Heand Wang, 2022). It has also been also shown that PSB can increase the available P
by reducing the intensity of soil P retention (Halder et al., 1990; Chen et al., 2006;
Ku et al., 2018; Tian et al., 2021),
Reportedly, PSB shows different effects on P transformation in different soils.
Hafeez et al. (2019) found that PSB raised the labile P fraction considerably in an
alkaline (pH 8.1) sandy loam soil after adding TCP. Delfim et al. (2020) found that
PSB exerted strong effects on NaOH-P in Andisol and Ultisol, which are bothacidic soils with similar P fractions. Furthermore, PSB showed an ability to slash“P-fixation capacity” (Biswas et al., 2022). The reports described above areinformative, but few comparative studies have examined effects of the same PSB
strains on P fractions in Lateritic red earths (La) and Cinnamon soils (Ci) withdifferent P fractions and PRP.
PSB can be divided into two categories according to their substrates: one isinorganic P bacteria that can convert insoluble inorganic P (such as inorganic
phosphate, phosphorous lime, etc.) into soluble P; One is organic P bacteria thatcan mineralize P containing organic substances such as phospholipids and organic
P pesticides. However, most P bacteria can not only dissolve inorganic P, but alsodegrade organic P, so it is difficult to accurately distinguish between organic P
bacteria and inorganic P bacteria. There are many bacteria that have been isolated
and confirmed to have phosphate solubilizing ability: Wani et al. (2005) isolated
Serratia, Pseudomonas and Bacillus with P-solubilizing ability from different
13rhizosphere soils; Chen et al. (2006) identified Serratia, PSB of genera Bacillus,
Rhodococcus,

Arthrobacter,

Chryseobacterium,

Delftia,

Gordonia

andPhyllobacterium; There are also Xanthomonas , Enterobacter, Pantoea, Klebsiella,Vibrioproteolyticus,

Xanthobacter

agilis,Burkholderia,

Paraburkholderia, Novosphingobium, and Ochrobactrum and so on (Chen et al.,2021; Chung et al. 2005; De Freitas et al. 1997; Vazquez et al. 2000).
PSB are widely distributed in the soil. PSB exist in normal soil environments
such as forests, farmlands, and orchards, and in adverse environments such as
mining areas, saline alkali lands, heavy metal contaminated areas andlow-temperature soils. The species and quantity of PSB in different habitats arealso different, and their distribution is affected by many factors, such as soil
properties, tillage measures and methods, rhizosphere effects, etc. The species anddiversity of PSB in the rhizosphere soil of natural forests are generally higher thanthose of artificial forests. The number and population abundance of PSB in the soilof phosphate mining areas are generally higher than those of heavy metal pollution
areas and saline areas. The abundance of PSB affected by soil organic components
and available P content.
The distribution of PSB is related to tillage measures and methods. The
physiological metabolic activities of different plant roots are different, so thecommunity structure and dominant structure of P bacteria in rhizosphere are also
different. In addition, cultivated crop intercropping, reasonable rotation and othertillage methods will affect the microbial community structure of soil P bacteria,
which will increase the number and abundance of P bacteria. Besides, the14

distribution of PSB also shows obvious rhizosphere effect.

1.1.2.2 Mechanism of PSB release P
The P release mechanism of PSB to insoluble inorganic P is relatively
complex. At present, the generally recognized mechanisms mainly include thesecretion of organic acids, the production of inorganic acids, the assimilation of
NH4+, the secretion of iron carriers and extracellular polysaccharides, etc.
P release by organic acids is a mechanism widely recognized by PSB. Organic
acids can not only reduce the pH in the environment, but also chelate with metal
cations in the environment to dissolve insoluble phosphates (Wang et al., 2009).
Different kinds of organic acids have different dissolution effects on insoluble
phosphates (Lu et al., 1998). PSBs isolated from the rhizosphere of Trifolium
repens can secrete various organic acids at different level, such as malic acid,
oxalic acid detected in the process of P release (Li et al., 2018).
The P release of most PSB is closely related to the low molecular weightorganic acids produced by their metabolism. On the one hand, these organic acids
can bind with cations such as Ca2+, Mg2+, Al3+, Fe3+, and release phosphate ions;
On the other hand, it can also reduce the environmental pH, thus promoting thedissolution of insoluble phosphate (Seshachala and Tallapragada, 2012). Many
studies have shown that the pH of the strain culture medium is significantly
negatively correlated with the total organic acid content, and the P release ability issignificantly correlated with the organic acid concentration and pH (Yang et al.,2021). The kind and content of organic acids secreted by different PSB are also15different. As mentioned above, such as gluconic acid, malic acid and oxalic acid
were detected.
In addition, gluconic acid is the main organic acid synthesized by PSB in theprocess of P release (Lin et al., 2006; Lee et al., 2012; Stephen and Jisha, 2011),
and gluconic acid is oxidized by glucose under the joint action of glucose
dehydrogenase (GDH) and coenzyme pyrroloquinoline quinone (PQQ) producedby bacteria. Therefore, GDH or PQQ synthesis related genes of PSB are crucial totheir P release function. PQQ biosynthesis involves many genes, often in the formof gene clusters (Wagh et al., 2014; Kim et al., 2003; Gliese et al., 2010).
Some PSB can produce HCl, HN3, H2SO4 and other inorganic acids to release
insoluble phosphates. Some bacteria of the genera Nitrosomonas spp. andThiobacillus spp. can produce nitric acid and sulfuric acid to dissolve phosphate
compounds. However, compared with organic acids, the P release efficiency of
inorganic acids is relatively low. In addition, H2S released by acidophilic sulfur
oxidizing bacteria can react with iron phosphate to produce ferrous sulfate, thus
releasing phosphate ions. Roy and Roy (2019) found that Delftia spp. can oxidize
thiosulfate and elemental sulfur to sulfate, and the accumulation of P in plants
increased significantly after inoculation of the strain.
PSB release proton hydrogen through the assimilation of NH4+, reducing theenvironmental pH, thus causing the dissolution of phosphate. Illmer and Schinner(1995) found that the pH of Pseudomonas spp. culture solution decreased
significantly, and the effective P content increased, but no organic acid was
detected. Therefore, they believe that the release of H+ accompanied by respiration16

or assimilation of NH4+ is another important P release mechanism.
The iron carrier secreted by PSB is a small molecule iron ion chelate, which
can combine with heavy metal ions in the soil to activate insoluble inorganic P. In
addition, the formed metal iron carrier complex can combine with the iron carrier
receptor protein on the cell membrane to enter cells, improve the iron absorption ofplants and promote plant growth. Similar to the chelation effect of iron carrier, theexopolysaccharide secreted by the PSB has abundant hydroxyl and carboxylic acidgroups on its surface, which can react with the Al3+, Zn2+, Fe3+, Mg2+ in the soil torelease phosphate ions. The strain secreting exopolysaccharide has stronger ability
to degrade P than the strain not secreting exopolysaccharide, and the strain
secreting exopolysaccharide and organic acid has stronger ability to degrade
phosphates.
It is generally believed that enzymatic hydrolysis is the main mechanism oforganic P degradation. Common enzymes include phosphatase, C-P lyase, phytase,etc. Phosphatase can remove the phosphate group in phosphate ester, which can begenerally divided into acid phosphatase and alkaline phosphatase. The content ofacid phosphatase was higher in acid soil; Alkaline phosphatase is common in
neutral and alkaline soils. C-P lyase can catalyze the cleavage of C-P bond, thereby
degrading organic P. Phytin is the main component of organic P in soil, it can’t beabsorbed by plants directly; phytase can degrade phytin into inositol and inorganic
phosphate. Therefore, P bacteria planted in plant rhizosphere can improve P
absorption of plants by secreting phytase.

17

1.1.2.3 What can PSB do for agriculture
The transformation of soil P by PSB can be described as that PSB promotes
the redistribution of P in soil through its own dissolution of insoluble phosphates.
The reason why it is called "redistribution" here is that: under the action of PSB,
the transformation result of soil P is not only the increase of soluble P, but also thecontent change of each extractable fractions. These changes combined thetransformation of insoluble phosphate into soluble phosphate and the re fixation orre adsorption of soluble phosphate by each extracted fraction. The relationships areplotted in the following diagram (Fig.1.1.2.3). PSB released its metabolites(organic acids, phosphatases, polysaccharides, etc.) into the soil solution, where
insoluble phosphates are dissolved or mineralized, releasing dissolved P into thesoil solution. Some of the dissolved P released into the soil solution is used by the
PSB for its own biomass synthesis, some is reabsorbed or immobilized by the soil,
and the remainder is retained in the soil solution as available P which can be useddirectly by plant.
PSB converts non-available P into effective P from the soil throughmineralization (secretion of phosphatase, phytase, etc.) or dissolution (secretion oforganic acids, protons, etc.), and there are many reports on PSB improving crop
yield and increasing P accumulation in crops (Alori et al., 2017), regarding theability to function under environmental stress There are many reports of PSBs that
dissolve P to promote growth (Dey et al., 2021). Therefore, using PSB to inoculate
soil is an effective strategy for soil P release, and it is a good way to reduce
chemical P fertilizer input and alleviate environmental pressure.
18

Fig. 1.1.2.3 Soil phosphorus translocation under the influence of PSB

19

1.2 Research purpose
The purpose of this study was to study the P stratification, enzyme activity
and organic acid dynamics induced by PSB in soil and rhizosphere soil, so as toshow the role of P solubilizing bacteria in the dynamic change of soil P structure,and to analyze the effect of PSB on P accumulation in maize seedlings based on
this. The effects of PSB on the bacterial community in the rhizosphere of maize
seedlings were studied by detecting and analyzing the changes of bacterial
community structure and diversity in the rhizosphere of maize seedlings caused by
PSB. In the soil inoculation and co culture experiments, this study used two kindsof soils with large differences in P grade structure to test, aiming to prompt thephysiological and biochemical differences of PSB in different soil environments(mainly different P composition environments), so as to evaluate the ability of PSB
as a P supply means under environmental conditions. By studying the influence of
PSB inoculation on the composition and diversity of rhizosphere bacterial
community of maize seedlings, it is analyzed that PSB affects plant growth byaffecting the structure or diversity of rhizosphere bacterial community, so as toevaluate the enthusiasm of PSB inoculation. The innovation of this study is to
explore for the first time the different effects of the same PSB strain on the Pclassification of laterite and cinnamon soil. In addition, this study also discussed
the difference of the degradation ability of PSB in the culture medium and soil, andrevealed the relationship between the soil type, P component change and PSB
strain.

0

1.3 Research frame

the effect of PSB on soils available phosphorus out put

the effect of PSB on maize seedling phosphorus accumulation

The role of phosphate solubilizing bacteria in soil
P dynamics and P accumulation of maize seedlings
the effect of PSB on maize rhizosphere soil bacterial community structure
Fig. 1.3.1. Research frame of my study

1

PSB isolation
Identification
Phosphate release ability
Viability in strilized soils
PSB for experiment

Incubate PSB with ster ilized soils
Soil P fractions
Soil organic acid
Soil phosphatase activity
Co culture PSB with maize
seedling in ster ilized soils
Soil P fractions
Soil organic acid
Soil phosphatase activity
Maize seedling P accumulation
Co culture PSB with maize
seedling in natur al soil
Rhizosphere soil 16S rDNA
Species
Alpha diversity
Beta diversity
Canonical correspondence

The role of PSB in maize seedling P accumulation

Fig. 1.3.2. Research flow of my study

2

analyseThe frame of the study was indicated in Fig.1.3.1. And the flow oht
the study was shown in Fig. 1.3.2. In the short term, this research started
with the isolation and identification of PSB, determining the identified
PSB strains’ P releasing ability to 5 phosphates in shake flasks, and their
viability in sterilized soils. PSB strains that exhibited a greater P releasecapacity and had viability in sterilized soils were used to investigate theeffect of PSB on soil P release and maize seedling P accumulation.

3

Chapter II

Isolation and Screening of PSB Strains

This chapter presents the procedure for isolating phosphatesolubilizing bacteria (PSB) from soil and subsequently screening themfor further experiments. The processes involved in this study encompass
the isolation of PSB from freshly collected natural soils, identification of
the isolated PSB strains, selection of different bacterial strains forphosphate release experiments using shake flasks, and testing thesurvival capability of strains exhibiting high phosphate release ability insoil. Following these experiments, five strains demonstrating both highphosphate release ability and the capacity to survive in sterilized La andCi soils were chosen for subsequent investigations.

2.1 Materials and Methods
2.1.1 PSB separation and identification
To isolate PSB, non-cultivated La and rhizosphere soil in which
Chinese cabbage (Brassica rapa L. var. pekinensis Rupr, Fig.2.1.1) had
been grown were used.

4

Fig.2.1.1 Brassica rapa L. var. pekinensis Rupr

2.1.1.1 PSB separation
One gram of each soil was suspended with 99 mL 0.85% NaClsterilized solution and was gradient diluted (Fig.2.1.2).
1g
10mL

10mL

10mL

99mL

90mL

90mL

90mL

10-2

10-3

10-4

10-5

100μL

100μL

Fig.2.1.2 Image of dilution scheme of soil suspension

5

100μL

After 100 µL quantities of 10-3, 10-4, and 10-5 dilutions were
cultured on NBRIP agar medium (Nautiyal, 1999), they were incubatedat 28°C for 5 days.
Select those colonies with earliertransparent ring formation (Fig.2.1.3),
wider ring width and higher ringtransparency

forpurification.

The

selected colonies were purified on
NBRIP (TCP source) until monoclonalcolonies were obtained.
Fig.2.1.3
Appearance
transparent ringsofFig.2.1.4 Image of PSB cultivation in LB

Each monoclonal colony was partly collected in 1.5 mL EP tubes
contained with 1 mL sterile pure water for DNA extraction for
identification and partly inoculated into 5 mL LB* which were
contained in 15 mL culture tube with dual position cap to amplify cells
for expanded cultivation (Fig.2.1.4).
6

2.1.1.2 PSB DNA extraction and identification
For the preliminary identification of 35 strains, DNA wereextracted via Chelex100.
Add 2 grams of Chelex100 into a 50 mL tube, filled pure water to
20 mL, and then put a magnetic stir-bar into the tube. Sterilized the tube
by autoclave at 121℃ for 15 min. After cooling, stirred the Chelex100
suspension on the magnetic stirrer. Aliquot 200 μL suspension into
sterile 0.5 mL EP tubes while keeping the suspension well mixed.
Remove 20 μL colony suspension from 1.5 mL EP tubes into 0.5 mL EP
tube which contained with chelex100 suspension, and then vortex forabout 10 seconds for mixing. Centrifuge the sample tubes quickly forabout one second so that all the samples were at bottom together. Put the
samples tubes into heat block at 95℃ for 15 min. Vortex and centrifuge
again. DNA were in the supernatant.
The kit was used to extract DNA of 5 selected PSB which wereused for the following experiments: Purified strains were enrichmentwith LB at 28℃ for 24 hours, then used to extract DNA with TIANamp
Bacteria DNA Kit (Tiangen biotech Co., Ltd, Beijing, China). Extracted
DNA were used as a temple for PCR amplification with universal
bacterial primers targeting the 16S rRNA gene which are 27F (Lane,
1991) and 1492R (Turner et al., 1999).
7

PCR was performed with 1μg template DNA, 1 μL (10 μM) of eachprimer, 12.5 μL 2×Taq PRC Mix (KT210, Tiangen biotech Co., Ltd,
Beijing, China), 9.5 μL double distilled H2O, and followed the step blow:
initial denaturation (94°C, 3 min), denaturation (94°C, 30 sec),
annealing (55℃, 30 sec), extension (72°C, 1 min). Final extension(72℃, 5 min) was performed after 30 cycles between denaturation andextension.
PCR products were used to agarose gel electrophoresis, thenstained the gels with GeneGreen Nucleic Acid Dye (RT210, Tiangenbiotech) and visualized by Gel imaging system (WD-9413B, Beijing
Liuyi Biotechnology Co., Ltd, China). Purified PRC products with
TIANgel Midi Purification Kit (DP209, Tiangen biotech). Purified
PCR-amplified 16S rDNA fragments were sequenced by AuGCT Co.,
Ltd, Wuhan, China. The obtained 16S rDNA sequences of isolated
strains were compared and upload to apply the NCBI number in NCBI
GenBank.

2.1.1.3 Strain Preservation
Incubated LB with PSB inoculation at 28℃ for 24 hr with rotary
shaking at 120 g. Transfer 200 micro liter culture and 300 micro liter
50% glycerol* to a 1.8 mL sterile cryo tube together. Mixed well thenstored in -20℃ for short time (half a year) storage and -80℃ for long8

term storage.

2.1.2 PSB pre screen
2.1.2.1 Shake flask culture
PSB strains were cultured respectively in LB liquid media at 28°C
for 24 hr. Culture solutions were centrifuged and then washed threetimes with 0.85% NaCl sterilized solution to collect the bacterial cells.
To resuspend the cells, P free NBRIP medium was used. The OD600 was
adjusted to 0.1 to obtain PSB suspensions for use in additional
experiments.
To test phosphate releasing ability, Ca3(PO4)2 (tricalcium phosphate,
TCP), FePO4, AlPO4, Phytin (inositol hexakisphosphate, Mg and Ca salt)
and lecithin (Yuanye, Shanghai, China) with the same P contents wereadded, respectively, to NBRIP as the P source. Adjusted the pH with 0.1
M NaOH and HCl to ensure that the pH reaches 7.0 ± 0.2 after autoclave.
Then the NBRIP was autoclaved at 115°C for 20 min. The PSB
suspension was inoculated into the sterilized NBRIP at a ratio of 1%(v/v) of the culture medium. Later, PSB was cultured with incubation at
28°C for 5 days. The rotation speed of 150 g min-1 was used with arotary shaker. After culture, the soluble inorganic P in NBRIP solution
content was measured.

9

2.1.2.2 P concentration of culture solution measure
Soluble inorganic P concentrations of all extracts and the digested
solution were quantified using Molybdenum blue method* (Murphy andRiley, 1962).

2.1.2.3 PSB soil viability test
For this step, the soils were autoclaved twice at 121°C for 60 min.
The viability of PSB in La and Ci was tested. After the PSB suspension(2.5 mL) was inoculated into 50 g soil, 12.5 mL sterile distilled water
was used to maintain the moisture level. Soils were incubated at 28°C in
the dark for 7 days.
After incubation, about 1 g soil was suspended with 99 mL
sterilized 0.85% NaCl solution. Furthermore, 50 μL of soil suspension
was spread on NBRIP agar medium and was cultured at 28°C for 5 days.
Strains that formed colonies with transparent circles on NBRIP wereconsidered to have soil viability.

2.1.3 PSB release P from phosphates in shake flask
2.1.3.1 Shake flask culture
PSB suspension was prepared as that in 2.1.2.1 whereas the culture
method also as same as that in 2.1.2.1. Each phosphate has a control
without PSB inoculation. Each culture and control had three replicates.
10

2.1.3.2 P concentration of culture solution measure
Same as 2.1.2.2.

2.1.3.3 pH of culture solution measure
The pH of culture solution was measured by pH meter*.

2.1.3.4 Organic acid of culture solution detection
Filtered the culture solution by a 0.45 μm filter membrane. Use thefiltered solution to measure the organic acid. This measurement was
carried out on the LC-100 HPLC instrument made by WuFeng Shanghai
China and using C18-AQ as the separation column.
Eight organic acids are used as the standard. They are oxalic acid,tartaric acid, malic acid, malonate acid, acetic acid, maleic acid, citricacid and succinic acid. Use 0.02M KH2PO4 solution of pH2.3 as themobile relative sample for analysis. Keep the flow rate at 1ml/min, andcontrol the column temperature of the separation column at 28 ℃. At
this time, the pressure in the column is relatively maintained at 11 MPa.
The organic acid mixture with gradient concentration is configured
for measurement, and the separation diagram is displayed in the results.
Make corresponding standard curve for organic acid.

11

2.1.4 Data analysis
Software (SPSS Statistics, Ver. 21.0.0.0; IBM Corp., Chicago, USA)was used to analyze the data.

2.2 Results
2.2.1 PSB identification
Thirty five PSB strains were initially isolated and identified as 6
genera (including Enterobacter, Klebsiella, Phytobacter, Erwinia,
Pantoea, and Pseudomonas) and 14 Top-hit taxon. They areEnterobacter sichuanensis, LECZ_s, Klebsiella huaxiensis, Phytobacter
ursingii, Erwinia persicina, Pantoea dispersa, Pantoea endophytica,
Pantoea rodasii, NEIG_s, Pseudomonas alloputida, Pseudomonas lactis,
Pseudomonasmonteilii,

Pseudomonas

plecoglossicida,andPseudomonas protegens (Table 2.2.1).
The list shows the similarity and completeness of the comparison
results. As the preliminary sequencing is one-way sequencing, thecompleteness is about 50%.
Fourteen PSB strains (A~N) which has the highest similarity ofeach top-hit strain were used for pre screen.

12

Table 2.2.1 Preliminary Identification
Name

Top-hit taxon

Top-hit strain

Similarity (%)

Completeness (%)

AEnterobacter sichuanensis

WCHECl1597

99.65

59.3

Z0

Enterobacter sichuanensis

WCHECl1597

99.54

59.2

H2

Enterobacter sichuanensis

WCHECl1597

99.39

56.4

N

LECZ_s

GN03164

99.32

50.1

F

Klebsiella huaxiensis

WCHKl090001(T)

96.44

44.5

C

Phytobacter ursingii

ATCC 27989

96.32

47.9

B50

Phytobacter ursingii

ATCC 27989

97.13

41.6

D

Erwinia persicina

NBRC 102418

99.63

55.8

B16

Erwinia persicina

NBRC 102418

99.64

57.5

B15

Erwinia persicina

NBRC 102418

99.55

45.5

B14

Erwinia persicina

NBRC 102418

99.7

45.2

B12

Erwinia persicina

NBRC 102418

99.54

44.4

B11

Erwinia persicina

NBRC 102418

99.65

59.1

B8

Erwinia persicina

NBRC 102418

99.86

48.1

B7

Erwinia persicina

NBRC 102418

99.65

59.2

B4

Erwinia persicina

NBRC 102418

98.15

59.2

B3

Erwinia persicina

NBRC 102418

99.30

58.4

13

Top-hit taxonomy
Bacteria;Proteobacteria;Gammaproteobacteria;Enterobacterales;Enterobacteriaceae;Enterobacter
Bacteria;Proteobacteria;Gammaproteobacteria;Enterobacterales;Enterobacteriaceae;Enterobacter
Bacteria;Proteobacteria;Gammaproteobacteria;Enterobacterales;Enterobacteriaceae;Enterobacter
Bacteria;Proteobacteria;Gammaproteobacteria;Enterobacterales;Enterobacteriaceae;Enterobacter
Bacteria;Proteobacteria;Gammaproteobacteria;Enterobacterales;Enterobacteriaceae;Klebsiella
Bacteria;Proteobacteria;Gammaproteobacteria;Enterobacterales;Enterobacteriaceae;Phytobacter
Bacteria;Proteobacteria;Gammaproteobacteria;Enterobacterales;Enterobacteriaceae;Phytobacter
Bacteria;Proteobacteria;Gammaproteobacteria;Enterobacterales;Erwiniaceae;Erwinia
Bacteria;Proteobacteria;Gammaproteobacteria;Enterobacterales;Erwiniaceae;Erwinia
Bacteria;Proteobacteria;Gammaproteobacteria;Enterobacterales;Erwiniaceae;Erwinia
Bacteria;Proteobacteria;Gammaproteobacteria;Enterobacterales;Erwiniaceae;Erwinia
Bacteria;Proteobacteria;Gammaproteobacteria;Enterobacterales;Erwiniaceae;Erwinia
Bacteria;Proteobacteria;Gammaproteobacteria;Enterobacterales;Erwiniaceae;Erwinia
Bacteria;Proteobacteria;Gammaproteobacteria;Enterobacterales;Erwiniaceae;Erwinia
Bacteria;Proteobacteria;Gammaproteobacteria;Enterobacterales;Erwiniaceae;Erwinia
Bacteria;Proteobacteria;Gammaproteobacteria;Enterobacterales;Erwiniaceae;Erwinia
Bacteria;Proteobacteria;Gammaproteobacteria;Enterobacterales;Erwiniaceae;Erwinia

B2

Erwinia persicina

NBRC 102418

98.95

58.8

B1

Erwinia persicina

NBRC 102418

98.98

67.3

M

Pantoea dispersa

LMG 2603

99.58

52.2

W7

Pantoea dispersa

LMG 2603

100

55.8

W6

Pantoea dispersa

LMG 2603

100

61.3

W4

Pantoea dispersa

LMG 2603

100

57.9

W3

Pantoea dispersa

LMG 2603

100

59.2

W2

Pantoea dispersa

LMG 2603

100

48.4

E

Pantoea endophytica

596

99.38

55.1

B

Pantoea rodasii

SHQLI9-1

99.72

58.9

G

NEIG_sR17(2017)

99.71

47.3

L

Pseudomonas alloputida

Kh7

100

53.6

K

Pseudomonas lactis

DSM 29167

99.85

46.7

B22

Pseudomonas lactis

DSM 29167

99.88

56.3

B21

Pseudomonas lactis

DSM 29167

99.59

66.2

J

Pseudomonas monteilii

NBRC 103158

99.89

63.4

I

Pseudomonas plecoglossicida

NBRC 103162

100

51.3

H

Pseudomonas protegens

CHA0

96.00

64.9

14

Bacteria;Proteobacteria;Gammaproteobacteria;Enterobacterales;Erwiniaceae;Erwinia
Bacteria;Proteobacteria;Gammaproteobacteria;Enterobacterales;Erwiniaceae;Erwinia
Bacteria;Proteobacteria;Gammaproteobacteria;Enterobacterales;Erwiniaceae;Pantoea
Bacteria;Proteobacteria;Gammaproteobacteria;Enterobacterales;Erwiniaceae;Pantoea
Bacteria;Proteobacteria;Gammaproteobacteria;Enterobacterales;Erwiniaceae;Pantoea
Bacteria;Proteobacteria;Gammaproteobacteria;Enterobacterales;Erwiniaceae;Pantoea
Bacteria;Proteobacteria;Gammaproteobacteria;Enterobacterales;Erwiniaceae;Pantoea
Bacteria;Proteobacteria;Gammaproteobacteria;Enterobacterales;Erwiniaceae;Pantoea
Bacteria;Proteobacteria;Gammaproteobacteria;Enterobacterales;Erwiniaceae;Pantoea
Bacteria;Proteobacteria;Gammaproteobacteria;Enterobacterales;Erwiniaceae;Pantoea
Bacteria;Proteobacteria;Gammaproteobacteria;Pseudomonad
ales;Pseudomonadaceae;Pseudomonas
Bacteria;Proteobacteria;Gammaproteobacteria;Pseudomonad
ales;Pseudomonadaceae;Pseudomonas
Bacteria;Proteobacteria;Gammaproteobacteria;Pseudomonad
ales;Pseudomonadaceae;Pseudomonas
Bacteria;Proteobacteria;Gammaproteobacteria;Pseudomonad
ales;Pseudomonadaceae;Pseudomonas
Bacteria;Proteobacteria;Gammaproteobacteria;Pseudomonad
ales;Pseudomonadaceae;Pseudomonas
Bacteria;Proteobacteria;Gammaproteobacteria;Pseudomonad
ales;Pseudomonadaceae;Pseudomonas
Bacteria;Proteobacteria;Gammaproteobacteria;Pseudomonad
ales;Pseudomonadaceae;Pseudomonas
Bacteria;Proteobacteria;Gammaproteobacteria;Pseudomonad
ales;Pseudomonadaceae;Pseudomonas

2.2.2 PSB pre screen
The first column in the Table 2.2.2 is the name of the strain (namedby author), and the second to sixth columns are marked with the name ofphosphate respectively. The data in the column indicated the soluble
inorganic P concentration of the shake flask solution after the PSB (in theleftmost column) was inoculated and cultured for 5 days. The seventh
column is the results of PSB survival tests in soils.
According to the measurement results of soluble inorganic P in
shake flask, it was found that strains F and G had released more P thanother strains from TCP; strains A and H released more P from FePO4; F,
H and A released more P from AlPO4; and B and M released more P from
Phytin. All of six PSB had almost not released P from Lecithin.
These 6 strains were incubated in sterilized La and Ci to test theirsurvival ability in sterilized soil environment. After one week incubating,the incubated soil was made into suspension and spread onto NBRIP agar
plates. These plates, coated with soil suspension, were incubated at 28 ℃.
After 5 days, colonies with transparent circles grew on the NBRIP agar
plates of soil samples inoculated with strains A, B, F, G, and H. No
colonies grew on the plates of soil samples inoculated with strain M. This
indicated that strains A, B, F, G, and H could survived and maintained
TCP solubility in sterilized La and Ci, while strain M may not have thisability or may require different nutrients to grow.
15

Table 2.2.2 PSB strains pre screen
Strain

TCP

FePO4

AlPO4

PhytinLecithin

A

0.257

0.015

0.013

0.306

0.001

B

0.265

0.002

0.010

0.448

0.000

C

0.286

0.001

0.000

0.303

0.000

D

0.188

0.010

0.005

0.113

0.000

E

0.212

0.003

0.008

0.162

0.000

F

0.483

0.005

0.015

0.389

0.000

G

0.594

0.003

0.012

0.381

0.000

H

0.181

0.018

0.013

0.096

0.000

I

0.220

0.006

0.005

0.313

0.001

J

0.162

0.012

0.003

0.223

0.000

K

0.405

0.005

0.002

0.137

0.000

L

0.162

0.002

0.011

0.159

0.000

M

0.383

0.003

0.001

0.392

0.000

N

0.441

0.002

0.003

0.228

0.000

Viability

○
○

○
○
○

×

The data is the content of soluble inorganic P in the shake flask solution, the unit is g/L; ○ : after 7
days of inoculation in the soil, it is found that it survives and maintains the P release ability
through testing, × indicates no viable bacteria are detected.

16

Table 2.2.3 Strain information of PSB used for following experiments
StrainA
B
F
G
H

SourceLateritic red earths
rhizosphere soil of Chinese
cabbagerhizosphere soil of Chinese
cabbagerhizosphere soil of Chinese
cabbage
Lateritic red earths

Closest relatives
Enterobacter
chuandaensis

Similarity

Pantoea rodasii

99.72

Klebsiella aerogenes

92.96

Pseudomonas
hunanensis
Pseudomonas
protegens

99.44

99.86
98.38

Classification
Bacteria;Proteobacteria;Gammaproteobacteria;Enterobacterales;Enterob
acteriaceae;Enterobacter
Bacteria;Proteobacteria;Gammaproteobacteria;Enterobacterales;Erwinia
ceae;Pantoea
Bacteria;Proteobacteria;Gammaproteobacteria;Enterobacterales;Enterob
acteriaceae;Klebsiella
Bacteria;Proteobacteria;Gammaproteobacteria;Pseudomonadales;Pseud
omonadaceae;Pseudomonas
Bacteria;Proteobacteria;Gammaproteobacteria;Pseudomonadales;Pseud
omonadaceae;Pseudomonas

NCBI number
ON778739
ON778745
ON7078779
ON778780
ON778778

The evolutionary history was inferred using the Neighbor-Joining method [1]. The optimal tree with the sum of
branch length = 0.34542664 is shown. The tree is drawn to scale, with branch lengths in the same units as those of theevolutionary distances used to infer the phylogenetic tree. The evolutionary distances were computed using theMaximum Composite Likelihood method [2] and are in the units of the number of base substitutions per site. The
analysis involved 10 nucleotide sequences. Codon positions included were 1st+2nd+3rd+Noncoding. All positions
containing gaps and missing data were eliminated. There were a total of 1361 positions in the final dataset.
Evolutionary analyses were conducted in MEGA7 [3].
1. Saitou N. and Nei M. (1987). The neighbor-joining method: A new method for reconstructing phylogenetic
trees. Molecular Biology and Evolution 4:406-425.
2. Tamura K., Nei M., and Kumar S. (2004). Prospects for inferring very large phylogenies by using theneighbor-joining method. Proceedings of the National Academy of Sciences (USA) 101:11030-11035.
3. Kumar S., Stecher G., and Tamura K. (2016). MEGA7: Molecular Evolutionary Genetics Analysis version 7.0

Fig. 2.2.3 Phylogenetic tree

for bigger datasets.Molecular Biology and Evolution 33:1870-1874.

17

Finally, 5 PSB strains (A, B, F, G and H) were selected for following
experiments.

2.2.3 Five PSB strains’ information
The 5 PSB that selected for the following experiments weresequenced with two-way sequencing. Their NCBI number showed inTable 2.2.3. Compare the sequence of bacteria species with that of similar
species to build the phylogenetic tree (Fig. 2.2.3).

2.2.4 PSB strains release P from phosphates in shake flask
2.2.4.1 Strain A release P from phosphates in shake flask
After 5 days, the P released from TCP by A is 0.27 g/L, and the pH
was 4.59, which is 2.44 lower than that of control 7.03 (Table 2.2.4.1a).
Compared with the dissolution of TCP, the dissolution of FePO4 by A wasvery weak. After 5 days, it was only 0.013 g/L. The pH dropped to 4.00,
1.30 lower than that of control 5.30, and less than that of TCP dissolution.
The ability of A to release P from AlPO4 was similar to that fromFePO4 (Table 2.2.4.1a). It released 0.012 g/L of P in 5 days, which was
the smallest release compared to the release of TCP and FePO4. However,the pH dropped from 6.40 to 3.37, which is the largest pH drop comparedto the release of TCP and FePO4.

18

Table 2.2.4.1a Inorganic phosphate release in shake flask withstrain A inoculation
TCP
FePO4
AlPO4
solution P
0.270
0.013
0.012
concentration (mg/L)
pH
4.59
4.00
3.37

The determination of TCP release and pH change of strain A for 5days showed that the pH decreased significantly on the first day and thesecond day of the 5 days, and then became flat on the third day(Fig.2.2.4.1a). The concentration of P in the solution rose rapidly on thefirst 4 days and became flat on the fifth day. It can be seen that whenstrain A releases TCP, pH was negatively related to the amount released.

Fig. 2.2.4.1a Strain A release P from TCP in 5 days

The release of organic phosphate by strain A in phytin shows good
release power (Table 2.2.4.1b). The amount of P released in 5 days is19higher than that in TCP, reaching 325 mg/L, and the pH drops to 4.24.
Strain A seems to have weak release effect on lecithin. 1 mg/L release isdetected, and the pH drops to 3.68.
Table 2.2.4.1b Organic phosphate release in shake flaskwith strain A inoculation
solution P concentration (mg/L)
pH

phytin325
4.24

lecithin
1.01
3.68

The determination of the release of phytin and the change of pH instrain A for 5 days showed that the trend of pH decrease was similar tothat of dissolved TCP, and it tended to be flat after the third day, while therelease of phytin showed a sharp increase in the first two days, and tended
to be flat after the second day (Fig. 2.2.4.1b).

Fig. 2.2.4.1b Strain A release P from Phytin in 5 days

20

The organic acid analysis of the culture medium 5 days after shaking
the flask showed that the output of organic acids is high in thephytin-source culture solution with acetic acid, malic acid, oxalic acid,and also trace succinic acid (Table 2.2.4). No known acid was found inlecithin-source culture solution.

Table 2.2.4 oganic acid in culture solutioin of shake flaskPstrainsource
TCP

B
F
G
H

Oxalic Tartaric Malic Malonate Acetic Maleic Citric Succinic SUM——————————————— mg/L ———————————————

112.5

FePO4

B
H

AlPO4

B
H

24.9
136.9

PhytinA
B
F
G
H

Lecithin

A
B
F
G
H

145.1
trace

45.4
596.4
882.8

trace

303.0
596.4
883.6

0.8

trace

330.0
284.1

17.6
8.5

trace

trace
233.5
288.7
28.0

trace

52.2

611.1
54.8

0.4
trace

95.5
633.7

1.4

trace

7.7

trace

0.3

24.9
0.3

21.0
21.7
10.2
12.3
13.7

100.3
365.1
368.2
391.9
701.1

trace

17.6
8.5
7.7

2.2.4.2 Strain B release P from phosphates in shake flask
For strain B, the release amount of TCP is the largest in 3 inorganic
phosphates as 290 mg/L, and the pH drops to 3.06 (Table 2.2.4.2a). Strain

21

B released about 1.80 mg/L FePO4, and the pH dropped to 3.15. The
release P by strain B from AlPO4 is higher than that from FePO4, reachingover 10 mg/L, and the pH dropped to 3.34.

Table 2.2.4.2a Inorganic phosphate release in shake flask with strain B inoculation
solution P concentration (mg/L)
pH

TCP

FePO4

AlPO4

290
3.06

1.80
3.15

10.15
3.34

When release inorganic phosphates, strain B secreted low variety
and quantity of organic acid (Table 2.2.4). No standard organic acid was
detected in TCP and FePO4 source culture solution. The organic acid in
AlPO4 source culture solution is mainly oxalic acid.
Release of phytin was higher than the release of inorganic phosphate,
reaching 459 mg/L, and the pH decreased to 5.33 (Table 2.2.4.2b). There
was almost no release of lecithin, even when the pH of the culture
medium decreased to 3.31.

Table 2.2.4.2b Organic phosphate release in shake flaskwith strain B inoculation
phytinlecithinsolution P concentration (mg/L)
pH

459
5.33

0.00
3.31

Strain B secretes different variety and quantities of organic acids
when releasing two organic phosphates (Table 2.2.4). When phytin was
22released, malic acid was the main component, acetic acid and succinicacid are also secreted, and several unknown acids were detected. When
lecithin was released, oxalic acid was mainly secreted, and malic acidwas also slightly secreted.

2.2.4.3 Strain F release P from phosphates in shake flask
Strain F can release the three inorganic phosphates provided (Table
2.2.4.3a). The release of TCP is higher than that of FePO4 and AlPO4,
reaching 477 mg/L, and the pH drops to 4.65. The release of FePO4 was
the lowest among the three, only 5.49 mg/L, and the pH decreased to 3.71.
The release of AlPO4 is slightly higher than that of FePO4, which is 15.22
mg/L, and the pH drops to 3.72.

Table 2.2.4.3a Inorganic phosphate release in shake flask with strain F inoculation
solution P concentration (mg/L)
pH

TCP

FePO4

AlPO4

477
4.65

5.49
3.71

15.22
3.72

It can be seen that when strain F releases TCP, it first continues toincrease the P concentration in the solution, and then decreases, while thepH first drops rapidly, and when the P concentration decreases, the pHraised. This indicates that strain F may consume the inorganic P in thesolution for its own biosynthesis, thus causing the decrease of P
concentration in the solution.
23

Strain F mainly secreted oxalic acid, malic acid, and acetic acid in
the process of dissolving TCP. In addition, an unknown acid was detected(Table 2.2.4).

Fig. 2.2.4.3a Strain F release P from TCP in 5 days

The release amount of F to phytin was 371 mg/L, and the pH value
reaches 3.83 (Table 2.2.4.3b). No release of lecithin from strain F was
detected, and the pH decreased to 3.58.

Table 2.2.4.3b Organic phosphate release in shake flask with strain F inoculation
phytin371
3.83

solution P concentration (mg/L)
pH

Lecithin
0.00
3.31

From the 5-day release dynamics of F to phytin, it is consistent with24

the release trend of F to TCP. The pH first drops sharply, then rises gently.
The amount of P released corresponds to the pH, and continues toincrease at the same time when the pH drops sharply, then decreases, andthen tends to be flat. It seems like that when F releases P, it produced aclimax of biosynthesis, thus consuming a large amount of inorganic P in
the environment.

Fig. 2.2.4.3a Strain F release P from phytin in 5 days

Strain F mainly secretes oxalic acid when releasing two kinds oforganic phosphates, but the amount secreted in phytin solution is muchgreater than that in lecithin solution (Table 2.2.4). In addition, strain F
also secretes malic acid and a small amount of succinic acid inphytin-source culture solution, and trace succinic acid in lecithin-source
culture solution.
25

2.2.4.4 Strain G release P from phosphates in shake flask
The release of G to TCP is 555 mg/L, and the pH drops to 4.56,
higher than that of the other two (Table 2.2.4.4a). The release of G to
FePO4 is low, only about 4.01 mg/L, and the pH drops to 4.06. The
release of AlPO4 from G is about 14.26 mg/L, and the pH drops to 4.08.

Table 2.2.4.4a Inorganic phosphate release in shake flask with strain G inoculation
solution P concentration (mg/L)
pH

TCP

FePO4

AlPO4

555
4.56

4.01
4.06

14.26
4.08

Strain G's release of P from TCP also has a rapid rise and then adecline process, and then a slow rise trend (Fig. 2.2.4.4a). The change ofpH decreases rapidly with the rapid increase of the concentration of P in
the solution. When the concentration of P decreases, the pH tends to beflat, and then when the concentration of P increases slowly, the pH also
rises slowly. Although strain G consumed inorganic P in the environment,
its release was still significantly higher than that of other bacteria.
Strain G secretes acetic acid and another unknown acid with highconcentration in the process of releasing TCP (Table 2.2).

26

Fig. 2.2.4.4a Strain G release P from TCP in 5 days

The release amount of strain G to phytin was 363 mg/L, and the pH
decreased to 3.96. The release of strain G to lecithin was also not detected,
but the pH also decreased to 3.9 (Table 2.2.4.4b).

Table 2.2.4.4b Organic phosphate release in shake flask with strain G inoculation
phytin363
3.96

solution P concentration mg/L
pH

Lecithin
0.00
3.90

The release dynamics are consistent with the trend towards TCP(Fig.2.2.4.4b). After the release peak, the P concentration in the solution
starts to decline, tends to be flat after one day, and then has an upward
trend. The pH decreases rapidly at the same time of rapid release, then27

tends to be flat, and basically remains unchanged after the next day. It is
speculated that G will also have a rapid production period of biosynthesis,
during which a large amount of inorganic P in the environment will be
consumed.

Fig. 2.2.4.4b Strain G release P from phytin in 5 days
Strain G mainly secreted oxalic acid, acetic acid and succinic acidwhen releasing phytin. Strain G was not detected to release, but traces oftartaric acid and acetic acid were still detected in lecithin-source culture
solution.

2.2.4.5 Strain H release P from phosphates in shake flask
Strain H only released 174 mg/L of TCP, but the pH decreased to4.30 (Table 2.2.4.5a). The release amount of H to FePO4 and AlPO4 is28

close, 14.58 mg/L and 13.15 mg/L respectively, and the pH drops to 3.8
and 3.67.

Table 2.2.4.5a Inorganic phosphate release in shake flask with strain H inoculation
solution P concentration (mg/L)
pH

TCP

FePO4

AlPO4

174
4.30

14.58
3.80

13.15
3.67

Strain H secretes a large amount of acetic acid when releasing TCP.
When releasing FePO4, no known organic acid was detected (Table 2.2.4).
When releasing AlPO4, trace amounts of acetic acid and succinic acid
were detected.
The release of organic phosphate by strain H was 94 mg/L in
phytin-source culture solution, and the pH value decreased to 4.59; Therelease of lecithin was 0.14 mg/L, and the pH decreased to 3.57 (Table
2.2.4.5b).

Table 2.2.4.5b Organic phosphate release in shake flask with strain H inoculation
solution P concentration (mg/L)
pH

phytinLecithin

94
4.59

0.14
3.57

When strain H releases the two organic acids, it secretes acetic acid
(Table 2.2.4). In addition, citric acid and succinic acid are also detected inphytin, and oxalic acid is mainly secreted in lecithin. But its secretion was
lower than that in phytin.

29

2.3 Discussion
This chapter introduces the method of obtaining PSB used in thisstudy. The difference in the release ability of the bacteria to three
insoluble inorganic phosphates and two organic phosphates was detected
through shake flask experiment. The survival ability of PSB in the soil
was verified through the culture experiment inoculated to sterile soil.
Finally, five PSB were selected as the strains used in this experiment.
When the five strains were inoculated in the soil, they had the survival
ability, and the release ability of the five strains to five kinds of phosphatein the shake flask was better than other strains.
As shown in Fig. 2.3.1, among the five strains, the largest TCP
release was strain G, followed by strain F; The release amount of FePO4is the largest for strain H, followed by strain A, which is 2.6-7.5 times
higher than strain B, F and G; strain F released the most AlPO4, and there
was only little difference among the five strains; The largest number ofPhytin releases is B; The weak release of lecithin is strain A. It can beseen that strains with strong ability to release inorganic phosphates do notnecessarily have a high release capacity for organic phosphates.

30

31

Figure 2.3.1 Soluble inorganic P concentration in culture solution
containing different P sources after 5 days incubation with 5 PSB isolates(A-H) or no bacterial isolate (Control). Different letters indicate
significant differences (p < 0.05; one-way ANOVA, Tukey, n = 3). Error
bars = SE.

When five strains released five kinds of phosphates, strain B caused
the maximum decrease of pH in inorganic phosphate TCP, FePO4, AlPO4and organic phosphate lecithin, but the P release was lower than otherstrains; When strain B releases phytin, the decrease of pH is the smallest,
but the release of P is the largest.

32

Figure 2.3.2 pH of culture solution after 5 days shake flask culture. The"*" symbol indicates significant differences (p < 0.05; one-way ANOVA,
Tukey, n = 3). Error bars = SE.

Taking pH as a covariate, univariate analysis was conducted on
strain, phosphate type, and solution P concentration (P release amount). It
was found that the strain had a significant impact on the type of P release(P < 0.05) when PSB released inorganic phosphate. However, inorganic P
sources did not have a significant impact on P release, and there was nointeractive impact between strain type and P source type on P release. The
influence of pH on the concentration of P in the solution was found to bestatistically significant (Table 2.3.1).

Table 2.3.1 Tests of Between-Subjects Effects
Between-Subjects Factors
Dependent Variable: P concentration
strains
phosphate
Source
P
A
TCP
pH
0.002
B
FePO4
PSB
0.042
F
AlPO4
phosphate
0.074
G
PSB * phosphate
0.198
33

H

When releasing organic phosphoric acid, the strain has a significant
effect on the limited amount of P released by species, while the type of P
source still has no significant effect on P release, but the strain and typeof P source have a significant interaction on P release. pH cannot
effectively reflect the concentration of P in solution (Table 2.3.2).

Table 2.3.2 Tests of Between-Subjects Effects
Between-Subjects Factors
Dependent Variable: P concentration
strains
phosphate
SourcepAphytinpH
0.647
Blecithin
PSB
0.011
F
phosphate
0.739
G
PSB * phosphate
0
H

Therefore, the decrease of pH has statistical significance for the
release of inorganic phosphate, but not for the release of organic
phosphate.
It can be seen from Table 2.2.4, when TCP was released, acetic acidwas secreted by strain F, G and H, and strain H secreted 882.8 mg/L;
oxalic acid and malic acid were found in F shake flask.
Limited organic acids were found in FePO4 and AlPO4 shake flask.
When phytin was released, oxalic acid was found in strain A, F and
G culture solution, while malic acid was found in all except for strain G.
Acetic acid was found in all except for strain F, and succinic acid was34

found in all strains culture solutions.

Also limited organic acids were found in lecithin shake flask.
Based on the types and total amount of organic acids detected, it
appeared that PSB secreted more types of organic acids when releasing
TCP and phytin compared to the other three phosphates, with a higher
total amount. This might be one of the reasons why PSB had a higher
release amount of TCP and phytin.

35

Chapter III

PSB release P from sterilized soils (inoculation experiment)
In this chapter, inoculation experiment was done. Five PSB strains(A, B, F, G and H) which screened from chapter 2 were inoculated into
La and Ci that with great different P fractions to observe the effect ofthem. The changes of soil P fractions, organic acid and phosphataseactivity in the soils were detected to evaluate the ability of PSB release P
of La and Ci.

3.1 Materials and methods
3.1.1 Materials and treatments
Two types of soils were used in this step (Table 3.1.1.1). Lateritic
red earths (La), from Nanning, Guangxi, China (22°50'28.6"N
108°11'25.7"E), and Cinnamon soils (Ci) from Fenyang, Shanxi, China(37°17'10.0"N

111°43'11.8"E).

Both

soils

are

collected

from

non-cultivated lands.

Table 3.1.1.1 Soils used in this experiment
Soil group

Abbr.

LocationpH

Lateritic red earths

La

Nanning, Guangxi, China (22°50'28.6"N 108°11'25.7"E)

5.5

Cinnamon soils

Ci

Fenyang, Shanxi, China (37°17'10.0"N 111°43'11.8"E)

8.0

36

La is acidic with pH 5.4, and the content of available P is low as 0.1
mg/kg soil; Ci is alkaline with pH 8.0, and available P content is higher
than La as 6.0 mg/kg soil. The total P content of the two is close, 0.6 and0.7 g/kg soil respectively.
Soils were air dried after removal of non-soil components such asstones and plant roots. The dried soils were crushed and sieved by 1 mmsieve and then autoclaved as that in 2.1.2.3 (PSB soil viability test).
Strain A, B, F, G and H were used for the reason that strain F and G
released more P from TCP, strain A and H released more P from AlPO4and FePO4, and B from phytin. And all of these 5 strains survived in thesterilized soil.
PSB suspension was prepared as that in 2.1.3.1 (Shake flask culture).
Inoculation experiments included three treatments (PSB treatment,
TCP treatment and combination treatment) and one control (Table
3.1.1.2). In the combination treatment, there were 5 experimental groups,
which were strain A, B, F, G and H. Each treatment (or each experimentalgroup) has three replicates.

37

Table 3.1.1.2 Treatments and conditions
Treatments

Soils and P
supplement

Inoculation

Moisture support

Control

La / Ci 50g

2.5 mL P free NBRIP

12.5 mL sterile distilled water

PSB Treatment

La / Ci 50g

2.5mL PSB suspensions

12.5 mL sterile distilled water

TCP Treatment

La / Ci 50g + 0.5g TCP

2.5 mL P free NBRIP

12.5 mL sterile distilled water

Combination Treatment

La / Ci 50g + 0.5g TCP

2.5mL PSB suspensions

12.5 mL sterile distilled water

P free NBRIP: NBRIP exclude TCP
PSB suspensions: A/B/F/G/H cells suspended with P free NBRIP to OD600 = 0.1
incubate: 28℃ 5days
Control: La-Ctrl, Ci-Ctrl
PSB treatment: LaA~LaH; CiA~CiH
TCP treatment: LaP, CiP
Combination treatment: LaAP~LaHP; CiAP~CiHPFor PSB treatment, 2.5 mL PSB suspensions were inoculated into 50
g sterilized soil respectively. Then 12.5 mL sterilized distilled water per
container was used to keep the moisture. For TCP treatment, 1% (w/w)
TCP was added to 50 g soil and mixed well, followed by autoclaving
twice at 121°C for 60 min. After cooling, 2.5 mL of P free NBRIP
medium and 12.5 mL sterilized distilled water were added. For
combination treatment, soils were prepared as that of TCP treatment.
After cooling, 2.5 mL PSB suspensions and 12.5 mL sterilized distilledwater were added into soils respectively. For use as a control, 2.5 mL of P
free NBRIP and 12.5 mL sterilized distilled water were added to 50 gsterilized soil. The three treatments and the control were cultured at 28°C
38

for 7 days. After incubation, about 0.1 g soil was suspended in 99 mL
normal saline and spread on NBRIP agar plate to confirm the survival of
the strain. One gram soil was air dried for measuring soil phosphataseactivity, and remaining soils were lyophilized for measuring P fractions
and organic acid content.

3.1.2 Soil P fractions measuring
Using the Hedley method, P fractions were determined with thefollowing procedure. First, 0.5 g dry soil was put into a 50 mL centrifuge
tube for extraction. Extraction was done using (1) about 5 cm² anionexchange resin (Selemion™ ion exchangeable resin; AGC Engineering
Co. Ltd., Chiba, Japan) and 30 mL distilled water; (2) 30 mL 0.5 M
NaHCO3; (3) 30 mL 0.1 M NaOH, and (4) 20 mL 1 M HCl, in sequence.
Each was conducted for 16 hr. Then, the resin was shaken with 20 mL 0.5
M HCl for 2 hr to extract resin P. Other extracts were NaHCO3-Pi,
NaOH-Pi and HCl-P. Then 5 mL NaHCO3 extract mixed with 10 mL 0.9
M H2SO4, and 0.5 g (NH4)2S2O8 were autoclaved at 120°C for 60 min to
obtain NaHCO3-P (both Pi and Po). Also, 5 mL NaOH extract, 10 mL 0.9
M H2SO4, and 0.6 g (NH4)2S2O8 were autoclaved at 120°C for 90 min to
obtain NaOH-PT. The difference between PT and the corresponding Pi is
Po. The P concentrations of all extracts and the digested solution were39

quantified using Molybdenum blue method as that in 2.1.2.2 (P content of
culture solution measure).

3.1.3 Soil pH measuring
The pH is measured in soil-water (1:2.5) by pH meter after
reciprocating vibration 30 min. The specific procedures are as follows:
weigh 10 g of dry soil and put into a 50 ml centrifuge tube, add 25 ml ofdistilled water into the tube, cover the tube and shake with a reciprocating
shaker at 120 g for 30 minutes. Leave the centrifuge tube standing for 1hrthen measure the pH of the upper solution with a pH meter.

3.1.4 Soil organic acid detection
Add 10 g dry soil to a centrifuge tube containing 10 mL of HPLC
mobile phase (here used 0.02 M KH2PO4 at pH 2.3). Reciprocating shakefor 2 h, keep still for 24 h, centrifuge at 8,000 x g for 5min, filter thesupernatant with a 4.5 μm filter.
HPLC analysis as same as 2.1.3.4 (Organic acid of culture solution
detection).

3.1.5 Soil phosphatase measuring
Soil phosphatase is a kind of enzyme that catalyzes themineralization of soil organic P compounds. Its activity directly affects
40

the decomposition, transformation and bioavailability of organic P in soil,
and is an indicator to evaluate the direction and intensity of soil P
biotransformation. Soil phosphatase was significantly affected by soil
carbon, nitrogen content, available P content and pH. Generally, there arethree types of phosphatase, alkaline, neutral and acidic, according to theiroptimum pH range.
This step used the Soil Phosphatase Activity Test Kit made by
Beijing Solarbio Science and Technology Co., Ltd. There were S-NP
(soil-neutral phosphatase) with Art. No. BC0460, S-AKP/ALP (soil-acid /alkaline phosphatase) with Art. No. BC0280 and S-ACP with Art. No.
BC0140.
The principle of these kits is that, in the corresponding pH
environment, phosphatase catalyzes the hydrolysis of disodium phenyl
phosphate to produce phenol and disodium hydrogen phosphate(Fig.3.1.5). The enzyme activity can be calculated by measuring theamount of phenol generated.
Phenyl Disodium Phosphate

Phosphatase

Phenol+Na2HPO
4

Phenol+2,6-Dibromobenzoquinone Chlorimid

corresponding pH Conditions

Indoxyl (660 nm)

Fig.3.1.5 Principle of enzyme assay
1) Fresh soil samples shall be dried in an oven at 37 ℃, and passthrough a 50 mesh sieve;
412) Weigh 0.1 g of air dried mixed soil, add 0.05 mL toluene, andshake gently for 15 min; Add 0.4 mL of reagent I and shake it well, place
it in 37 ℃ for constant temperature culture, and catalyze the reaction for
24h. After that time, quickly add 1mL Reagent II and fully mix it to stopthe enzyme catalyzed reaction. Centrifuge at 10,000 x g at room
temperature for 10 min, take the supernatant and place it on ice formeasuring.
3) Adjust the wavelength of the spectrophotometer to 660 nm, andadjust the distilled water to zero.
4) Add liquid into 1 mL glass cuvette according to the dosage in the
Table 3.1.5.

Table 3.1.5 Composition of solution for measurement of enzyme
activities
Reagent/μL
Ⅰ
Ⅲ
Ⅳ
Distilled waterStandard
Sample

Blank
50
100
20
830

Standard

Sample

100
20
830
50

100
20
830
50

Evenly mix and let stand at room temperature for 30 min. Measure
the absorbance at 660 nm (expressed as strain A).
5) Calculation

42

Enzyme activity：At 37 ℃, 1 nmol phenol is released per gram ofsoil per day as one enzyme activity unit.
Enzyme activity（U/g soil）=[C standard × (A sample - A blank) ÷ (A standard - A blank)] × VT
÷ W ÷ T × 1000
=725 × (A sample - A blank) ÷ (A standard - A blank) ÷ W
C standard: 0.5 μmol/mL; VT: Total volume of catalytic system,
1.45 mL; W: Soil sample weight, g; T: Catalytic reaction time, 24 h=1 d;
1,000: unit conversion factor, 1 μmol=1,000 nmol.

3.2 Results
3.2.1 Strain A release P from soils
After inoculation of strain A to La (LaA), four inorganic fractions
were significantly changed, while the organic fractions were not affected.
Inoculation to La with TCP (LaAP) caused significant changes in all
fractions relative to LaP (Table 3.2.1a).
After inoculation to Ci (CiA), all fractions except NaOH-Pi weresignificantly changed; After inoculation to Ci added with TCP (CiAP),the fraction except NaOH-Pi and HCl-P changes significantly comparedwith CiP (Table 3.2.1b).
Inoculation of strain A in both soils resulted in a decrease in pH (Fig.
3.2.1a). When the content of calcium phosphate in the soil is high (LaAP,43

CiA, CiAP), the oxalic acid in the soil is largely consumed (Fig. 3.2.1b).
In all treatments, LaAP had the largest pH decline rate and the largest
oxalic acid consumption rate.

44

Table 3.2.1a Soil fractions (g-P kg-1 soil) of strain A inoculation experiment in LaGroup
La-Ctrl
LaA
LaP
LaAP

Resin P
0.057±0.003
0.075±0.003**
0.468±0.016**
1.051±0.022**

NaHCO3-Pi
0.005±0.002
0.029±0.004**
0.263±0.008**
0.605±0.009**

NaHCO3-Po
0.205±0.007
0.210±0.005
0.212±0.006
0.288±0.006**

NaOH-Pi
14.29±0.24
15.55±0.36**
34.00±0.38**
43.00±0.32**

NaOH-Po
9.41±0.24
9.84±0.28
9.43±0.23
10.78±0.19**

HCl-P
0.0729±0.0108
0.0124±0.0032**
452±6**
432±4**

Values represent the mean ± SE (standard errors).
The significance was analyzed using an Independent-Samples T test. The significance of the LaA and LaP’s fractions is calculated relative to La-Ctrl, while thesignificance of the LaAP’s fractions is calculated relative to LaP.
**: p ＜ 0.01

Table 3.2.1b Soil fractions (g-P kg-1 soil) of strain A inoculation experiment in Ci
Group
Ci-Ctrl
CiA
CiP
CiAP

Resin P
1.76±0.01
3.05±0.01**
2.02±0.02**
4.10±0.01**

NaHCO3-Pi
4.13±0.01
4.49±0.04**
5.62±0.02**
6.17±0.02**

NaHCO3-Po
0.95±0.01
1.44±0.02**
0.94±0.03
1.43±0.02**

NaOH-Pi2.38±0.01
2.39±0.01
2.38±0.02
2.44±0.05

NaOH-Po
2.76±0.01
3.41±0.01**
2.76±0.03
3.69±0.01**

HCl-P
307±2
300±3**
1800±9**1787±7**

Values represent the mean ± SE (standard errors).
The significance was analyzed using an Independent-Samples T test. The significance of the CiA and CiP’s fractions is calculated relative to La-Ctrl, while thesignificance of the CiAP’s fractions is calculated relative to CiP.
**: p ＜ 0.01

45

Fig. 3.2.1a Soil pH of strain A inoculation experiment. The percentages
indicated on LaA, LaP, and LaAP represent the percentage of pH change
relative to La-Ctrl in each treatment; The percentages indicated on CiA,CiP, and CiAP represent the percentage of pH change relative to Ci-Ctrl
in each treatment.

Fig. 3.2.1b Soil organic acid (mg kg-1 soil) of strain A inoculation
experiment. The value "0" represents trace amounts. The terms used in
the context are as follows: "Succinic" refers to succinic acid, "Citric"
refers to citric acid, "Maleic" refers to maleic acid, "Acetic" refers toacetic acid, "Malonate" refers to malonic acid, "Malic" refers to malic
acid, and "Oxalic" refers to oxalic acid.
The activity of acid phosphatase (ACPase) in both soils were higher
than that of neutral phosphatase (NPase) and alkaline phosphatase46

(AKPase). The 3 phosphatase activities of all treatments of La werehigher than those of all treatments of Ci (Fig.3.2.1c). Inoculation of A
increased the activity of 3 phosphatase; Among them, the increase rate of
ACPase and NPase in La is greater than that in Ci (Table.3.2.1c).

Fig. 3.2.1c Phosphatase activity of strain A inoculation experiment
Table.3.2.1c Change rate (%) of phosphatase activity of strain A inoculation
experiment
ACPase NPase AKPase
ACPase NPase AKPase
LaA
32
25
9
CiA
15
14
27
LaP
-5
1
-1
CiP
-48
10
2
LaAP
49
19
41
CiAP
31
16
27
Change rate is relative to control (La -Ctrl and Ci-Ctrl).
＞ 0: increased, ＜ 0: decreased.

Pearson correlation analysis was conducted on the fractions in two
soils that had changed significantly in each treatment, and the factors thatcaused the change were found out from the five factors of soil pH, oxalic
acid consumption, and three phosphatase activities.
It was found that NPase had a significant positive effect on resin P in
La; pH had a significant negative effect on NaHCO3-Pi, while AKPase
activity had a positive effect. The significant change of other fractions
47

had no significant relationship with these five factors.
In La added with TCP, compared with LaP, all fractions had changed
significantly. Five factors had significant effects on resin P, NaHCO3-P
and NaOH-Pi, among which pH was negatively correlated and the otherfour were positively correlated. NaOH-Po was not affected by pH, and
HCl-P change was not caused by these five factors (Table.3.2.1d).
Among Ci and Ci added with TCP, five factors had significant
effects on resin, NaHCO3-Po and NaOH-Po, among which pH was
negatively correlated and the rests were positively correlated.
Furthermore, in Ci added with TCP, the reason for the significant increaseof NaHCO3-Pi was significantly related to the increase of oxalic acidconsumption, ACPase and AKPase activity (Table.3.2.1d).
Strain A showed different phosphatase activity release and oxalic
acid consumption in four treatments of two soils, and caused a decreasein pH. The changes of P fraction caused by inoculation had different
correlations with pH, oxalic acid consumption and phosphatase activity inthe four treatments, but generally showed negative correlation with pH
and positive correlation with other factors.

48

Table.3.2.1d Pearson correlation between soil indicators and soil P fractions in strain A inoculation experimentsoil pH

Oxalicconsume

ACPaseNPase

AKPasesoil pH

Oxalicconsume

La
Resin PNaHCO3-Pi
NaHCO3-Po
NaOH-Pi
NaOH-Po
HCl-P-0.860*-0.811*----0.936**-0.938**-0.876*-0.940**-0.998**
0.978**
0.920**
0.934**
0.942**-

0.998**
0.972**
0.911*
0.924**
0.945**-NPase

AKPase0.986**
0.989**
0.971**-

0.998**
0.997**
0.984**-

0.866*
0.817*
0.906*-

0.988**
0.953**
0.992**
0.928**-

Ci
0.862*-

0.889*--0.882*-0.893*-0.930**-1.000**
1.000**
0.990**-

La+TCP
Resin PNaHCO3-Pi
NaHCO3-Po
NaOH-Pi
NaOH-Po
HCl-P

ACPase0.904*
0.911*
0.958**
Ci+TCP

0.994**
0.964**
0.889*
0.919**
0.923**-

0.995**
0.985**
0.938**
0.944**
0.941**-**: p ＜ 0.01, *: p＜0.05, -: p≥0.05-*: Significant negative correlation, *:Significant positive correlation

49-0.908*-0.934**-0.863*-0.999**
0.963**
0.987**
0.963**-

0.998**
0.965**
0.985**
0.965**-

3.2.2 Strain B release P from soils
Inoculation of strain B to La caused significant changes in four
inorganic fractions and NaHCO3-Po. After inoculation to La added withTCP (LaBP), compared with LaP, NaHCO3-P and NaOH-P changed
significantly (Table 3.2.2a).
After being inoculated with Ci (CiB), strain B caused significant
changes in fractions other than NaOH-P; After inoculation to Ci addedwith TCP (CiBP), compared with CiP, B caused a significant change inthe removal of three fractions, and NaHCO3-Po decreased significantly(Table 3.2.2b).
Inoculation of B caused a slight decrease in pH in the soil without
TCP, but a slight increase in the soil with TCP (Fig. 3.2.2a). At the sametime, when the content of calcium phosphate in the soil is high (LaBP,CiB, CiBP), the oxalic acid in the soil is largely consumed. Among all
treatments, LaBP has the highest oxalic acid consumption rate (Fig.
3.2.2b).

Fig. 3.2.2a Soil pH of strain B inoculation experiment. The percentages
indicated on LaA, LaP, and LaAP represent the percentage of pH change
relative to La-Ctrl in each treatment; The percentages indicated on CiA,CiP, and CiAP represent the percentage of pH change relative to Ci-Ctrl
50

in each treatment.

51

Table 3.2.2a Soil fractions (g-P kg-1 soil) of strain B inoculation experiment in LaGroup
La-Ctrl
LaB
LaP
LaBP

Resin P
0.057±0.003
0.084±0.005**
0.468±0.016**
0.457±0.012

NaHCO3-Pi
0.005±0.002
0.070±0.008**
0.263±0.008**
0.490±0.019**

NaHCO3-Po
0.205±0.007
0.333±0.009**
0.212±0.006
0.280±0.005**

NaOH-Pi
14.29±0.24
19.96±0.39**
34.00±0.38**
40.65±0.22**

NaOH-Po
9.41±0.24
9.09±0.31
9.43±0.23
11.39±0.23**

HCl-P
0.0729±0.0108
0.0007±0.0005**452±6**
437±9

Values represent the mean ± SE (standard errors).
The significance was analyzed using an Independent-Samples T test. The significance of the LaA and LaP’s fractions is calculated relative to La-Ctrl, while thesignificance of the LaAP’s fractions is calculated relative to LaP.
**: p ＜ 0.01，*: p ＜ 0.05.

Table 3.2.2b Soil fractions (g-P kg-1 soil) of strain B inoculation experiment in Ci
Group
Ci-Ctrl
CiB
CiP
CiBP

Resin P
1.76±0.01
3.98±0.01**
2.02±0.02**
4.10±0.01**

NaHCO3-Pi
4.13±0.01
4.36±0.05**
5.62±0.02**
5.82±0.06

NaHCO3-Po
0.95±0.01
1.54±0.01**
0.94±0.03
0.72±0.02**

NaOH-Pi2.38±0.01
2.38±0.01
2.38±0.02
2.49±0.04*

NaOH-Po
2.76±0.01
2.73±0.02
2.76±0.03
2.80±0.02

HCl-P
307±2
299±2**
1800±9**1790±6

Values represent the mean ± SE (standard errors).
The significance was analyzed using an Independent-Samples T test. The significance of the CiA and CiP’s fractions is calculated relative to Ci-Ctrl, while thesignificance of the CiAP’s fractions is calculated relative to CiP.
**: p ＜ 0.01，*: p ＜ 0.05.

52

Fig. 3.2.2b Soil organic acid (mg kg-1 soil) of strain B inoculation
experiment. The value "0" represents trace amounts. The terms used in
the context are as follows: "Succinic" refers to succinic acid, "Citric"
refers to citric acid, "Maleic" refers to maleic acid, "Acetic" refers toacetic acid, "Malonate" refers to malonic acid, "Malic" refers to malic
acid, and "Oxalic" refers to oxalic acid.

The activity of ACPase in both soils was higher than that of NPaseand AKPase. The 3 phosphatase activities of all treatments in La werehigher than those of all treatments in Ci. Inoculation of B increased theactivity of 3 phosphatase; Among them, the increase rate of NPase andAKPase in La is greater than that in Ci, but the increase rate of ACPaseactivity in Ci is greater than that in La (Fig. 3.2.2c).

53

Fig. 3.2.2c Phosphatase activity of strain B inoculation experiment

Table 3.2.1c Change rate (%) of phosphatase activity of strain B inoculation
experiment
ACPase NPase AKPase
ACPase NPase
AKPase
LaB
127
219
156
CiB
166
67
24
LaP
-5
1
-1
CiP
-48
10
2
LaBP
153
134
58
CiBP
183
90
29
Change rate is relative to control (La or Ci).
＞ 0: increased, ＜ 0: decreased.

Pearson correlation analysis was conducted on the fractions in two
soils that had changed significantly in each treatment, and the factors thatcaused the change were found out from the five factors of soil pH, oxalic
acid consumption, and three phosphatase activities.
It was found that the increases of resin P, NaHCO3-Po and NaOH-Pi
in La were related to the increase of oxalic acid consumption, ACPaseand NPase activity; The factors affecting the increase of NaHCO3-Pi andthe decrease of HCl-P were not among the five factors.
In La added with TCP, the four fractions of NaHCO3-P and NaOH-P
have changed significantly compared with LaP. The increase of oxalic
acid consumption and the activity of 3 phosphatase had significant
positive effects on the four fractions; PH is positively correlated with54

NaHCO3-Po and NaOH-P.
In Ci and Ci with TCP added, five factors have significant effects on
resin P and NaHCO3-Po. Among them, pH is negatively correlated in Ci
and positively correlated with resin P in Ci with TCP added; The change
of NaHCO3-Po was significantly decreased, and its change wasnegatively correlated with five factors.

55

Table 3.2.2d Pearson correlation between soil indicators and soil P fractions in strain B inoculation experimentsoil pH
Resin PNaHCO3-Pi
NaHCO3-Po
NaOH-Pi
NaOH-Po
HCl-P

0.889*-

Oxalicconsume0.850*
0.928**
0.929**-0.878*-

ACPaseLa0.853*
0.929**
0.928**-0.880*-NPase
0.848*
0.931**
0.932**-0.882*-

AKPase-soil pH-0.941**-0.931**-

Oxalicconsume0.999**
0.998**-

La+TCP
Resin PNaHCO3-Pi
NaHCO3-Po
NaOH-Pi
NaOH-Po
HCl-P

0.850*
0.879*
0.900*-

0.821*
0.935**
0.928**
0.950**-

0.813*
0.933**
0.928**
0.948**-

ACPaseNPase

AKPaseCi
0.999**
0.997**-

1.000**
0.999**-

0.997**
0.996**-

1.000**-0.957**-0.989**-0.920**-Ci+TCP
0.811*
0.933**
0.928**
0.946**-

0.831*
0.940**
0.935**
0.948**-**: p ＜ 0.01, *: p ＜ 0.05, -: p ≥ 0.05-*: Significant negative correlation, *: Significant positive correlation

56

0.989**-0.941**-0.999**-0.948**-0.999**-0.949**-Strain B also showed different phosphatase activity release andoxalic acid consumption in four treatments of two soils, and caused adecrease in soil pH in the soil environment without TCP, while it caused arise in pH in the environment with TCP. The changes of P fraction caused
by inoculation were correlated with the five factors, and there weresignificant differences among the four treatments.

3.2.3 Strain F release P of soils
After inoculation of strain F to La (LaF), four inorganic fractions
were significantly changed, while the organic fractions were not affected.
After inoculation to La with TCP (LaFP), only NaOH-Pi changed
significantly compared with LaP (Table 3.2.3a).
After inoculation to Ci (CiF), all fractions except HCl-P weresignificantly changed, in which NaOH-Pi was significantly decreased.
After inoculation to Ci added with TCP (CiFP), the fraction except
NaOH-Pi and HCl-P increased significantly compared with CiP (Table
3.2.3b).
After inoculation to Ci (CiF), all fractions except HCl-P weresignificantly changed, in which NaOH-Pi was significantly decreased.
After inoculation to Ci added with TCP (CiFP), the fraction except
NaOH-Pi and HCl-P increased significantly compared with CiP(Table.3.2.3b).
57

Table 3.2.3a Soil fractions (g-P kg-1 soil) of strain F inoculation experiment in LaGroup
La-Ctrl
LaF
LaP
LaFP

Resin P
0.057±0.003
0.079±0.003**
0.468±0.016**
0.506±0.016

NaHCO3-Pi
0.005±0.002
0.043±0.004**
0.263±0.008**
0.266±0.007

NaHCO3-Po
0.205±0.007
0.210±0.005
0.212±0.006
0.216±0.009

NaOH-Pi
14.29±0.24
15.44±0.25**
34.00±0.38**
35.36±0.29**

NaOH-Po
9.41±0.24
9.56±0.26
9.43±0.23
9.56±0.27

HCl-P
0.0729±0.0108
0.0121±0.0033**
452±6**
448±8

Values represent the mean ± SE (standard errors).
The significance was analyzed using an Independent-Samples T test. The significance of the LaA and LaP’s fractions is calculated relative to La-Ctrl, while thesignificance of the LaAP’s fractions is calculated relative to LaP.
** p ＜ 0.01，* p ＜ 0.05

Table 3.2.3b Soil fractions (g-P kg-1 soil) of strain F inoculation experiment in Ci
Group
Ci-Ctrl
CiF
CiP
CiFP

Resin P
1.76±0.01
2.39±0.01**
2.02±0.02**
2.82±0.07**

NaHCO3-Pi
4.13±0.01
5.84±0.11**
5.62±0.02**
6.17±0.06**

NaHCO3-Po
0.95±0.01
1.78±0.05**
0.94±0.03
1.78±0.06**

NaOH-Pi2.38±0.01
2.28±0.02**
2.38±0.02
2.36±0.03

NaOH-Po
2.76±0.01
2.83±0.02**
2.76±0.03
3.01±0.02**

HCl-P
307±2
300±4
1800±9**1792±9

Values represent the mean ± SE (standard errors).
The significance was analyzed using an Independent-Samples T test. The significance of the CiA and CiP’s fractions is calculated relative to Ci-Ctrl, while thesignificance of the CiAP’s fractions is calculated relative to CiP.
**: p ＜ 0.01，*: p ＜ 0.05.

58

F inoculation only caused a slight decrease in pH in La, but caused aslight increase in the soil added with TCP (Fig.3.2.3a). When the content
of calcium phosphate in the soil is high (LaFP, CiF, CiFP), the oxalic acidin the soil is largely consumed. Among all treatments, CiF has the highest
oxalic acid consumption rate (Fig.3.2.3b).
The activity of ACPase in both soils was higher than that of NPaseand AKPase. The ACPase and AKPase activities of all treatments of La
were higher than those of all treatments of Ci (Fig.3.2.3c). The
inoculation of F increased the activity of AKPase in La, but decreased theactivity of AKPase in Ci. The increase rate of AKPase in La is greaterthan that in Ci (Table 3.2.3c).

Fig. 3.2.3a Soil pH of strain F inoculation experiment. The percentages
indicated on LaA, LaP, and LaAP represent the percentage of pH change
relative to La-Ctrl in each treatment; The percentages indicated on CiA,CiP, and CiAP represent the percentage of pH change relative to Ci-Ctrl
in each treatment.

59

Fig. 3.2.3b Soil organic acid (mg kg-1 soil) of strain F inoculation
experiment. The value "0" represents trace amounts. The terms used in
the context are as follows: "Succinic" refers to succinic acid, "Citric"
refers to citric acid, "Maleic" refers to maleic acid, "Acetic" refers toacetic acid, "Malonate" refers to malonic acid, "Malic" refers to malic
acid, and "Oxalic" refers to oxalic acid.

Fig. 3.2.3c Phosphatase activity of strain F inoculation experiment
Table 3.2.3c Change rate (%) of phosphatase activity of strain F inoculation
experiment
ACPaseNPase
AKPaseACPaseNPase
AKPase
LaF
4
6
0
CiF
-10
22
16
LaP
-5
1
-1
CiP
-48
10
2
LaFP
33
16
8
CiFP
-24
15
17
Change rate is relative to control (La or Ci).
＞ 0: increased, ＜ 0: decreased.

60

Pearson correlation analysis was conducted on the fractions in two
soils that had changed significantly in each treatment, and the factors thatcaused the change were found out from the five factors of soil pH, oxalic
acid consumption, and three phosphatase activities.
It was found that in La, resin P increased due to the decrease of pH
and the increase of oxalic acid consumption, and NaHCO3-Pi increased
due to the decrease of pH and the increase of oxalic acid consumption, as
well as the increase of acid and NPase activities; NaOH-Pi is positively
affected by NPase activities.
In La added with TCP, no significant factors related to the change of
NaOH-Pi were found.
In Ci and Ci added with TCP, pH shows a positive correlation, while
oxalic acid consumption still shows a positive correlation. ACPaseactivity shows a positive correlation in Ci with TCP, and a negative
correlation in Ci without TCP.
Strain F also showed different phosphatase activity release andoxalic acid consumption under four treatments of two soils. Inoculation
caused pH rise and oxalic acid consumption of other three treatments
except LaF. The changes of P fraction caused by inoculation werecorrelated with the five factors, and there were significant differencesamong the four treatments.
61

Table 3.2.3d Pearson correlation between soil indicators and soil P fractions in strain F inoculation experimentsoil pH

Oxalicconsume

ACPaseNPase

AKPasesoil pH

Oxalicconsume

La
Resin PNaHCO3-Pi
NaHCO3-Po
NaOH-Pi
NaOH-Po
HCl-P

Npase

AKPaseCi-0.867*0.867*----

0.999**-0.927**

0.993**

0.978**-0.896*0.887*

0.865*

0.909*-

0.837*

0.883*-

0.858*

0.860*

-

-

-

-

-

0.944**

0.938**-0.906*

0.937**

0.959**---

0.826*

-

-

-

-

-

-

-

-

-

-

-0.849*

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

La+TCP
Resin PNaHCO3-Pi
NaHCO3-Po
NaOH-Pi
NaOH-Po
HCl-P

ACPaseCi+TCP

-

-

-

-

-

0.991**

0.982**

0.983**-

0.956**

-

-

-

-

-

0.856*-

0.816*

-

-

-

-

-

-

-

0.933**

0.937**

0.916*-

0.893*

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

0.831*

-

-

-

-

-

-

-

-

-

-

-

**: p ＜ 0.01, *: p ＜ 0.05, -: p ≥ 0.05-*: Significant negative correlation, *:Significant positive correlation

623.2.4 Strain G release P of soils
After strain G was inoculated to La (LaG), significant changes in
NaHCO3-Pi and NaOH-Pi were caused, and the organic fractions were
not affected. After inoculation to La with TCP (LaGP), the fraction except
NaOH-Pi and HCl-P increased significantly compared with LaP (Table
3.2.4a).
After inoculation with Ci (CiG), the fraction increased significantly,
except NaOH-Po and HCl-P. After inoculation to Ci with TCP (CiGP),the fraction increased significantly compared with CiP, except HCl-P(Table 3.2.4b).
Inoculation of strain G caused a decrease in pH of La and Ci without
TCP, but caused a slight increase in pH of soil with TCP (Fig. 3.2.4a).
The consumption of soil oxalic acid was greater in the combination
treatment of the two soils (Fig. 3.2.4b).

Fig. 3.2.4a Soil pH of strain G inoculation experiment. The percentages
indicated on LaA, LaP, and LaAP represent the percentage of pH change
relative to La-Ctrl in each treatment; The percentages indicated on CiA,CiP, and CiAP represent the percentage of pH change relative to Ci-Ctrl
in each treatment.

63

Table 3.2.4a Soil fractions (g-P kg-1 soil) of strain G inoculation experiment in LaGroup
La-Ctrl
LaG
LaP
LaGP

Resin P
0.057±0.003
0.062±0.007
0.468±0.016**
0.542±0.009**

NaHCO3-Pi
0.005±0.002
0.056±0.005**
0.263±0.008**
0.335±0.013**

NaHCO3-Po
0.205±0.007
0.210±0.005
0.212±0.006
0.284±0.006**

NaOH-Pi
14.29±0.24
16.77±0.35**
34.00±0.38**
34.86±0.30

NaOH-Po
9.41±0.24
9.66±0.35
9.43±0.23
12.68±0.22**

HCl-P
0.0729±0.0108
0.0017±0.0008**
452±6**
451±6

Values represent the mean ± SE (standard errors).
The significance was analyzed using an Independent-Samples T test. The significance of the LaA and LaP’s fractions is calculated relative to La-Ctrl, while thesignificance of the LaAP’s fractions is calculated relative to LaP.
**: p ＜ 0.01，*: p ＜ 0.05.

Table 3.2.4b Soil fractions (g-P kg-1 soil) of strain G inoculation experiment in Ci
Group
Ci-Ctrl
CiG
CiP
CiGP

Resin P
1.76±0.01
1.92±0.01**
2.02±0.02**
2.84±0.02**

NaHCO3-Pi
4.13±0.01
4.97±0.07**
5.62±0.02**
5.03±0.03**

NaHCO3-Po
0.95±0.01
1.34±0.11**
0.94±0.03
1.36±0.09**

NaOH-Pi2.38±0.01
2.44±0.02*
2.38±0.02
2.56±0.03**

NaOH-Po
2.76±0.01
2.74±0.01
2.76±0.03
3.38±0.04**

HCl-P
307±2
304±3
1800±9**1793±9

Values represent the mean ± SE (standard errors).
The significance was analyzed using an Independent-Samples T test. The significance of the CiA and CiP’s fractions is calculated relative to Ci-Ctrl, while thesignificance of the CiAP’s fractions is calculated relative to CiP.
**: p ＜ 0.01，*: p ＜ 0.05.

64Fig. 3.2.4b Soil organic acid (mg kg-1 soil) of strain G inoculation
experiment. The value “0” represents trace amounts. The terms used in
the context are as follows: “Succinic” refers to succinic acid, “Citric”
refers to citric acid, “Maleic” refers to maleic acid, “Acetic” refers toacetic acid, “Malonate” refers to malonic acid, “Malic” refers to malic
acid, and “Oxalic” refers to oxalic acid.

The activities of ACPase in both soils were higher than that of NPaseand AKPase (Fig. 3.2.4c). The ACPase activity of LaGP was much higher
than that of the other three treatments, with an increase rate at 370%
(Table 3.2.4c).

Fig. 3.2.4c Phosphatase activity of strain G inoculation experiment

65

Table 3.2.4c Change rate (%) of phosphatase activity of strain G inoculation
experiment
ACPase NPase AKPase
LaG
8
3
9
CiG
LaP
-5
1
-1
CiP
LaGP
370
9
41
CiGP
Change rate is relative to control (La or Ci).
＞ 0: increased, ＜ 0: decreased.

ACPase
310
-48
152

NPase
19
10
23

AKPase73
252

As Table 3.2.4d showed, it was found that in La, NaHCO3-Pi and
NaOH-Pi were negatively correlated with pH, and positively correlatedwith oxalic acid consumption and phosphatase activity. None of the fivefactors was significant reasons causing changes in HCl-P.
In La added with TCP, the correlation of these five factors with thechange of each fraction is positive.
In Ci, NaHCO3-Pi and NaOH-Pi were negatively correlated with pH,
and positively correlated with oxalic acid consumption and phosphataseactivity.
In Ci with TCP added, the five factors all had positive effects on
resin, while they had negative effects on NaHCO3-Pi (NaHCO3-Pi of
CiGP is significantly reduced compared with CiP).
Strain G also showed different phosphatase activity release andoxalic acid consumption under four treatments of two soils, and caused adecrease in soil pH in the soil environment without TCP, while caused arise in pH in the environment with TCP. The changes of P fraction caused
by inoculation were related to all the five factors, but there were different
among the four treatments.
66

Table.3.2.4d Pearson correlation between soil indicators and soil P fractions in strain G inoculation experimentsoil pH

Oxalicconsume

ACPaseNPase

AKPasesoil pH

Oxalicconsume

La
Resin PNaHCO3-Pi
NaHCO3-Po
NaOH-Pi
NaOH-Po
HCl-P-0.945**-0.882*-0.917**
0.892*-

0.870*
0.951**-

0.842*-

0.935**
0.945**
0.991**-

0.936**
0.944**
0.991**-NPase

AKPase0.901*
0.895*-

0.928**
0.836*-

0.968**-0.841*-0.989**-0.909*-Ci
0.826*-

0.865*
0.898*--0.924**-0.931**-

0.953**
0.841*-

La+TCP
Resin PNaHCO3-Pi
NaHCO3-Po
NaOH-Pi
NaOH-Po
HCl-P

ACPase0.949**
0.848*
Ci+TCP

0.837*
0.864*
0.932**-

0.915*
0.937**
0.979**-**: p ＜ 0.01, *: p ＜ 0.05, -: p ≥ 0.05-*: Significant negative correlation, *:Significant positive correlation

67

0.997**-0.861*0.820*-

0.994**-0.922**0.828*-

0.994**-0.918**0.824*-

3.2.5 Strain H release P of soils
Table 3.2.5a Soil fractions (g-P kg-1 soil) of strain H inoculation experimentGroup

Resin PNaHCO3-Pi

NaHCO3-Po

NaOH-Pi

NaOH-Po

HCl-P

La-Ctrl
LaH

0.057±0.003
0.069±0.023

0.005±0.002
0.011±0.002

0.205±0.007
0.224±0.006

14.29±0.24
16.62±0.27**

9.41±0.24
9.57±0.38

0.0729±0.0108
0.0210±0.0049**

LaP
LaHP

0.468±0.016**
0.445±0.009

0.263±0.008**
0.456±0.015**

0.212±0.006
0.350±0.008**

34.00±0.38**
34.03±0.33

9.43±0.23
12.71±0.32**452±6**
443±9

Values represent the mean ± SE (standard errors).

The significance was analyzed using an Independent-Samples T test. The significance of the LaA and LaP’s fractions is calculated relative to La-Ctrl, while thesignificance of the LaAP’s fractions is calculated relative to LaP.
**: p ＜ 0.01，*: p ＜ 0.05.

Table 3.2.5b Soil fractions (g-P kg-1 soil) of strain H inoculation experiment in Ci
Group

Resin PNaHCO3-Pi

NaHCO3-Po

NaOH-Pi

NaOH-Po

HCl-P

Ci

1.76±0.01

4.13±0.01

0.95±0.01

2.38±0.01

2.76±0.01

307±2

CiH

2.43±0.01**

4.89±0.05**

1.12±0.02**

2.52±0.01**

2.87±0.01**

302±3

CiP

2.02±0.02**

5.62±0.02**

0.94±0.03

2.38±0.02

2.76±0.03

1800±9**CiHP

2.84±0.01**

6.05±0.14a**1.12±0.07*

2.53±0.04**

2.99±0.01**

1793±10

Values represent the mean ± SE (standard errors).

The significance was analyzed using an Independent-Samples T test. The significance of the CiA and CiP’s fractions is calculated relative to Ci-Ctrl, while thesignificance of the CiAP’s fractions is calculated relative to CiP.
**: p ＜ 0.01，*: p ＜ 0.05.

68Inoculation of strain H to La (LaH) caused significant changes in
NaOH-Pi and HCl-P fractions. Inoculation to La added with TCP (LaHP)
caused significant changes in NaHCO3-P and NaOH-Po relative to LaP(Table 3.2.5a).
After inoculation to Ci and C with TCP (CiH, CiHP), all fractions
except HCl-P were significantly changed (Table 3.2.5b).
The pH of four treatments in both soils decreased due to H
inoculation (Fig.3.2.5a). At the same time, when the content of calcium
phosphate in the soil is high (LaHP, CiH, CiHP), the oxalic acid in thesoil is largely consumed (Fig.3.2.5b).

Fig. 3.2.5a Soil pH of strain H inoculation experiment. The percentages
indicated on LaA, LaP, and LaAP represent the percentage of pH change
relative to La-Ctrl in each treatment; The percentages indicated on CiA,CiP, and CiAP represent the percentage of pH change relative to Ci-Ctrl
in each treatment.

69

Fig. 3.2.5b Soil organic acid (mg kg-1 soil) of strain H inoculation
experiment. The value "0" represents trace amounts. The terms used in
the context are as follows: "Succinic" refers to succinic acid, "Citric"
refers to citric acid, "Maleic" refers to maleic acid, "Acetic" refers toacetic acid, "Malonate" refers to malonic acid, "Malic" refers to malic
acid, and "Oxalic" refers to oxalic acid.
The activity of ACPase in both soils was higher than that of NPaseand AKPase. The ACPase of La was higher than that of Ci. Comparedwith the control (La, Ci), the content and increase of three phosphatasesof LaHP are higher than that of CiHP.

Fig. 3.2.5c Phosphatase activity of strain H inoculation experiment70

Table 3.2.5c Change rate (%) of phosphatase activity of strain H inoculation
experiment
ACPase NPase AKPase
ACPase NPase
AKPase
LaH
40
22
15
CiH
41
16
33
LaP
-5
1
-1
CiP
-48
10
2
LaHP
84
38
95
CiHP
44
18
22
Change rate is relative to control (La or Ci).
＞ 0: increased, ＜ 0: decreased.

From the Table 3.2.5d, it can be found that pH was negatively
correlated with fractions in the four inoculation treatments.
In La, oxalic acid consumption and acid phosphatase activity are
positively correlated with NaOH-Pi.
In La added with TCP, oxalic acid consumption and phosphataseactivity are positively correlated with NaHCO3-P and NaOH-Po.
In Ci, resin, NaHCO3-P and NaOH-Po were significantly correlatedwith the five factors; In Ci with TCP added, only resinP is related to thefive factors.
Strain H showed different phosphatase activity release and oxalic
acid consumption in four treatments of two soils, but both caused thedecrease of pH value. The changes of P fractionation caused byinoculation had different correlations with pH, oxalic acid consumption
and phosphatase activity in the four treatments, but generally showednegative correlation with pH and positive correlation with other factors.
71

Table 3.2.5d Pearson correlation between soil indicators and soil P fractions in strain H inoculation experimentsoil pH

Oxalicconsume

ACPaseNPase

AKPasesoil pH

Oxalicconsume

La
Resin PNaHCO3-Pi
NaHCO3-Po
NaOH-Pi
NaOH-Po
HCl-P-0.851*-0.863*--0.889*-0.910*
0.972**
0.991**-

0.878*-

0.907*
0.969**
0.992**-NPase

AKPase0.987**
0.861*
0.921**
0.857*-

0.954**
0.889*
0.992**
0.853*-

0.904*-

0.974**-

Ci---0.993**-0.872*-0.905*-0.906*-

0.993**
0.889*
0.952**
0.910*-

La+TCP
Resin PNaHCO3-Pi
NaHCO3-Po
NaOH-Pi
NaOH-Po
HCl-P

ACPase0.990**
0.922**
0.939**
0.941**
Ci+TCP

0.908*
0.970**
0.991**-

0.911*
0.972**
0.990**-**: p ＜ 0.01, *: p ＜ 0.05, -: p ≥ 0.05-*: Significant negative correlation, *:Significant positive correlation

72-0.992**-0.999**-

0.997**-

3.3 Discussion
This chapter introduces the experiment of PSB on the change of soil
P fractions. Five PSB strains with higher ability to release 3 kinds ofinsoluble inorganic phosphate and 2 kinds of organic phosphate in shakeflask culture were inoculated into two soils. The two soils are La and Ci,
which have very different P fractions. For P fractionation of two soils,
excluding residual P, P in La is mainly concentrated in NaOH extractablefraction, while P content distributed in other levels is very low. Due to thehigh content of iron and aluminum, La has a great potential to retain P; on
the other hand, La provide little calcium phosphate for PSB to release, sowhether PSB can change the P fractionation of La is studied in thisexperiment. Another kind of soil is Ci from Shanxi Province, China. Thissoil is weakly alkaline, with a high content of HCl extractable P, low ironand aluminum content. The content of NaOH extractable P is much lower
than that of La. Compared with La, it is easier for PSB to release HCl
extractable P.
According to the pH change data presented in Figure 3.3.1, the fivestrains tested caused a greater range of pH changes in La (in both PSB
and combined treatments) compared to Ci. In all treatments, strains A and
H caused a decrease in pH in La. Notably, H resulted in the lowest pH
values across all four La series (with strain A being the same as strain H
in the LaP series). On the other hand, strain B had the highest pH values73

in two La's treatments, while strain F had the highest pH values in two Ci's treatments. Furthermore, the addition of TCP led
to an increase in pH levels in LaP as well as in LaBP, LaFP, CiBP, CiFP, and CiGP, when compared to their respective
controls.

Fig. 3.3.1 Treatments’ pH of inoculation experiment

The pH changes caused by PSB in soil inoculation experiments were found to be inconsistent with the pH changes

74

observed in shake flask experiments. For example, strain B caused lower pH levels in TCP, FePO4, and AlPO4 in shake75

flask experiments than other strains, but it led to higher pH levels in soil
inoculation experiments than some other strains. These results suggestthat the pH responses of PSB vary depending on the environment.
From the perspective of soil organic acid detection (Table 3.3.1),
oxalic acid was detected in all soil samples. The amount of oxalic aciddetected in non-inoculated soil samples was higher than in samples
inoculated with PSB. The content of oxalic acid was nearly the same intreatments without TCP supply and with TCP supply. TCP supply does
not appear to alter the composition and content of organic acids in La soil
without inoculation, but it slightly affects the composition of some Ci
soil.
In addition to oxalic acid, malic acid was detected in uninoculated
La soil with a content of 1.9-3.3 mg/kg soil, and in uninoculated Ci soil
with a content of 9.3-9.4 mg/kg soil. Furthermore, malic acid, malonicacid, acetic acid, and citric acid were detected in the control group of Ci
soil.
In the PSB treatment of La soil, the inoculation of the strain slightly
reduced the content of oxalic acid in the soil, resulting in a reduction of
7.2-24.2 mg/kg soil. Malic acid was detected in all experimental groups,
and malonic acid was detected in all experimental groups except group A.
Group A also exhibited trace amounts of acetic acid.
In the combined treatment of La soil, the reduction in oxalic acid76

caused by inoculation ranged from 186 to 411 mg/kg soil, with the largest
reduction caused by strain H. Strain H secreted 0.6 mg/kg soil of malic
acid. Malic acid and malonic acid were detected in all experimentalgroups, while trace amounts of maleic acid were detected in group A.
In the PSB treatment of Ci soil, the reduction in oxalic acid caused
by inoculation ranged from 264 to 701 mg/kg soil, with the largest
reduction caused by strain H. The content of both malonic acid and acetic
acid secreted by all experimental groups was higher than in the control
group, with malonic acid being 5.8-56.6 mg/kg higher and acetic acidbeing 2.5-41 mg/kg higher than in the control soil. The citric acidsecreted by strain A, F, and H was 3.9-6.7 mg/kg higher than in thecontrol soil. Additionally, strains F and H secreted 7.5 mg/kg and 0.8
mg/kg of tartaric acid, respectively.
In the combined treatment of Ci soil, the reduction in oxalic acidcaused by inoculation ranged from 186 to 733 mg/kg soil, with the largest
reduction caused by strain H. In addition to oxalic acid, strain A secreted
40.6 mg/kg soil of acetic acid and 8.7 mg/kg soil of malic acid, strain B
secreted 41.0 mg/kg soil of malonic acid and 11.4 mg/kg soil of citricacid, strain F secreted 114.2 mg/kg soil of maleic acid and 7.0 mg/kg soilof tartaric acid, and strain H secreted 32.9 mg/kg soil of malonic acid and2.5 mg/kg soil of citric acid. Trace amounts of maleic acid were detected
in all experimental groups except group A.
77

Overall, the type and content of organic acids secreted by the samestrain in Ci soil are generally higher than in La soil. For soils with highcalcium phosphate content such as LaP, Ci, and CiP, the degree of changein oxalic acid is much greater than in La soil with low calcium phosphate
content. It was also observed that strain H secreted malic acid in LaP,
resulting in the largest reduction of oxalic acid. In Ci soil, the total
amount of other organic acids secreted by strains H and F was 118.2 and118.3, respectively, with their oxalic acid being reduced to 45.7 and 49.8,
much higher than the reduction observed in group A, B and G.

78

Table 3.3.1 Soil organic acid (mg kg-1 soil) of inoculation experimentgroup
La-Ctrl
LaA
LaB
LaF
LaG
LaH

Oxalic
439.0
406.7
438.2
425.4
436.0
421.7

Tartaric

Malic
1.9
trace
trace
trace
trace
trace

LaP
LaAP
LaBP
LaFP
LaGP
LaHP

452.6
64.4
161.3
59.9
266.9
41.2

3.3
trace
trace
trace
trace
0.6

Ci-Ctrl
CiA
CiB
CiF
CiG
CiH

746.8
213.8
482.5
49.7
661.5
45.7

9.3

CiP
CiAP
CiBP
CiFP
CiGP
CiHP

744.3
296.6
558.6
63.1
424.4
11.7

7.5
0.8

1.4

Malonate

Acetic
trace
trace

trace
trace
trace
trace

trace
trace

trace
trace
trace
trace
trace
0.9
52.2
35.8
53.7
6.7
57.5

9.4
7.0

trace

trace

trace

41.0
114.2
trace
32.9

79

Maleictrace

CitricSuccinic

455.9
64.4
161.3
59.9
266.9
41.8

trace

0.2
41.2
24.2
36.0
2.7
37.4

0.4
trace
trace
0.1

40.6
trace

8.7
trace
trace
trace
trace

trace

SUM
440.9
406.7
438.2
425.4
436.0
421.7

14.4
183
21.1
4.8
21.1

11.4
2.5

trace

772.0
325.5
542.5
168.0
675.7
163.9
753.7
345.9
611.0
184.3
424.4
47.1

Based on an analysis of soil phosphatase activity, La soil generally
exhibits higher levels of phosphatase activity compared to Ci soil.
ACPase activity was found to be the highest among the three types ofphosphatase. Additionally, all strains except F caused the increase ofphosphatase activity in all treatments of both soils.

Fig.3.3.2 Soil phosphatase activity (U/g soil/ day) of inoculation
experiment
While the application of PSB resulted in increased soil phosphataseactivities, the magnitude of the changes varied depending on the strain
80species, soil type, and treatment. Specifically, strains A, B, F, and H
consistently displayed increased ACPase activity across all four
treatments in both soil types. However, the enzyme activity caused bystrain G differed significantly across the four treatments. NPase activity
remained relatively stable in both La and Ci soils, while AKPase activity
showed irregular fluctuations.
In La inoculation experiment (Table 3.3.2a), compared with thecontrol (La-Ctrl), all groups of the PSB treatment were found to havesignificantly increased NaOH-Pi and decreased HCl-P. The TCP
fertilization changed the size of the inorganic P fractions (i.e. resin P,
NaHCO3-Pi, NaOH-Pi, and HCl-P), but organic P fractions (NaHCO3-Po,
NaOH-Po) remained similar. Compared with the TCP treatment (LaP), allgroups of the combination treatment, except LaFP, increased significantlyin NaHCO3-Pi, NaHCO3-Po and NaOH-Po; LaAP, LaBP and LaFP
increased significantly in NaOH-Pi. The interaction between PSB
inoculation and TCP supply was found to be significant for all P fractions
except HCl-P. Table 3.3.2b showed the changes of labile P, moderately
labile P and HCl-P in La inoculation experiment. La-Ctrl’s moderately
labile P (23.71 mg-P kg-1 soil) is much higher than that of labile P (0.27
mg-P kg-1 soil). When TCP was added, the increase of moderately labile P(19.72 mg-P kg-1 soil) was greater than that of labile P (0.67 mg-P kg-1
soil). When PSB were inoculated, the increases of moderately labile P
81were also greater than those of labile P in both PSB and combination
treatments.
For Ci inoculation experiment (Table 3.3.3a), compared with Ci-Ctrl,
all groups of the PSB treatment were found to have significantly
increased resin P and NaHCO3-Pi. TCP supply was found to havesignificantly different resin P, NaHCO3-Pi, and HCl-P, but organic P
fractions were unaffected, which is a similar finding to that for LaP.
Compared with the CiP, all groups of the combination treatment were
found to have significantly increased resin P. The interaction between
PSB inoculation and TCP supply was found to be significant for resin P,
NaHCO3-Pi, NaHCO3-Po, and NaOH-Po, whereas NaOH-Pi and HCl-P
were unaffected by it. As Table 3.3.3b shows, Ci-Ctrl’s labile P andmoderately labile P were similar in their pool size: 6.84 and 5.13 mg-P
kg-1 soil. TCP supply increased labile P by 1.74 mg kg-1 soil but noincrease in moderately labile P was detected. When PSB were inoculated,
the increases of labile P were greater or slightly greater than those ofmoderately labile P in both PSB and combination treatments, except
CiGP.
Pearson correlation analysis of the two soils with TCP supply (TCPand combined treatment) and without TCP supply (control and PSB
treatment) showed no significant correlation between labile P and HCl-P(data not shown).
82

Table 3.3.2a Soil P fractions (mg-P kg-1 soil) in La inoculation experimentGroup
La-Ctrl

Resin P
0.057±0.003

NaHCO3-Pi
0.005±0.002

NaHCO3-Po
0.205±0.007

NaOH-Pi
14.29±0.24

NaOH-Po
9.41±0.24

HCl-P
0.0729±0.0108

LaA
LaB
LaF
LaG
LaH

0.075±0.003
0.084±0.005
0.079±0.003
0.062±0.007
0.069±0.023

0.029±0.004*
0.070±0.008*
0.043±0.004*
0.056±0.005*
0.011±0.002

0.210±0.005
0.333±0.009*
0.210±0.005
0.210±0.005
0.224±0.006

15.55±0.36*
19.96±0.39*
15.44±0.25*
16.77±0.35*
16.62±0.27*

9.84±0.28
9.09±0.31
9.56±0.26
9.66±0.35
9.57±0.38

0.0124±0.0032*
0.0007±0.0005*
0.0121±0.0033*
0.0017±0.0008*
0.0210±0.0049*

LaP

0.468±0.016*

0.263±0.008*

0.212±0.006

34.00±0.38*

9.43±0.23452±6*

LaAP
LaBP
LaFP
LaGP
LaHP

1.051±0.022*
0.457±0.012
0.506±0.016
0.542±0.009*
0.445±0.009

0.605±0.009*
0.490±0.019*
0.266±0.007
0.335±0.013*
0.456±0.015*

0.288±0.006*
0.280±0.005*
0.216±0.009
0.284±0.006*
0.350±0.008*

43.00±0.32*
40.65±0.22*
35.36±0.29*
34.86±0.30
34.03±0.33

10.78±0.19*
11.39±0.23*
9.56±0.27
12.68±0.22*
12.71±0.32*

432±4
437±9
448±8
451±6
443±9

PSB
TCP
PSB * TCP

***
***
***

***
***
***

***
***
***

***
***
***

***
***
***

***
-

Values represent the mean of three replicates ± SE (standard errors).
Significant differences among means were tested with one-way ANOVA (Dunnett. Use La-Ctrl as control category for LaA ~LaH and LaP; use LaP for LaAP ~LaHP).
La-Ctrl: control, La with P-free NBRIP adding; LaA ~ LaH: PSB treatment, La with A ~ H suspensions inoculation; LaP: TCP treatment, La with TCP supply andP-free NBRIP adding; LaAP~LaHP: Combination treatment, La with A~H suspensions inoculation and TCP supply.
PSB, TCP, and PSB*TCP: Two-way ANOVA for the factors PSB inoculation, TCP supply, and the interaction of PSB inoculation ×TCP supply.
***: p < 0.001, **: p < 0.01, *: p < 0.05, −: p ≧ 0.05.

83

Table 3.3.2b Changes of labile P, moderately labile P and HCl-P in La inoculation experiment (mg-P kg-1 soil)
grouplabile P

moderately
labile P

relative to La
labile P Increase

La-Ctrl

0.27

LaA
LaB
LaF
LaG
LaH

0.31
0.49
0.33
0.33
0.30

0.05
0.22
0.06
0.06
0.03

LaP

0.94

0.67

LaAP
LaBP
LaFP
LaGP
LaHP

1.94
1.23
0.99
1.16
1.25

1.68
0.96
0.72
0.89
0.98

relative to La
moderately labile P
Increase

HCl-P decrease
23.71
0.06
0.07
0.06
0.07
0.05
relative to LaP
labile P Increase
HCl-P decrease
1.00
20.00
0.29
14.63
0.05
4.26
0.22
1.11
0.31
9.26

25.39
29.05
25.00
26.43
26.20

1.69
5.34
1.29
2.73
2.49

43.43

19.72

53.78
52.04
44.92
47.54
46.75

30.07
28.33
21.21
23.83
23.04

La-Ctrl control; LaA ~ LaH PSB treatment, La with A ~ H strains inoculating；LaP TCP treatment, La with TCP supply；LaAP ~ LaHP Combination treatment, La
with A~H strains inoculating and TCP supply；

84

Table 3.3.3a Soil P fractions (mg-P kg-1 soil) in Ci inoculation experimentGroup
Ci-Ctrl

Resin P
1.76±0.01

NaHCO3-Pi
4.13±0.01

NaHCO3-Po
0.95±0.01

NaOH-Pi2.38±0.01

NaOH-Po
2.76±0.01

HCl-P
307±2

CiA
CiB
CiF
CiG
CiH

3.05±0.01*
3.98±0.01*
2.39±0.01*
1.92±0.01*
2.43±0.01*

4.49±0.04*
4.36±0.05*
5.84±0.11*
4.97±0.07*
4.89±0.05*

1.44±0.02*
1.54±0.01*
1.78±0.05*
1.34±0.11*
1.12±0.02

2.39±0.01
2.38±0.01
2.28±0.02*
2.44±0.02*
2.52±0.01*

3.41±0.01*
2.73±0.02
2.83±0.02*
2.74±0.01
2.87±0.01*

300±3
299±2
300±4
304±3
302±3

CiP

2.02±0.02*

5.62±0.02*

0.94±0.03

2.38±0.02

2.76±0.03

1800±9*

CiAP
CiBP
CiFP
CiGP
CiHP

4.10±0.01*
4.10±0.01*
2.82±0.07*
2.84±0.02*
2.84±0.01*

6.17±0.02*
5.82±0.06
6.17±0.06*
5.03±0.03*
6.05±0.14*

1.43±0.02*
0.72±0.02*
1.78±0.06*
1.36±0.09*
1.12±0.07

2.44±0.05
2.49±0.04
2.36±0.03
2.56±0.03*
2.53±0.04*

3.69±0.01*
2.80±0.02
3.01±0.02*
3.38±0.04*
2.99±0.01*

1787±71790±61792±91793±91793±10PSBTCPPSB * TCP

***
***
***

***
***
***

***
***
***

***
***
-

***
***
***

***
-

Values represent the mean of three replicates ± SE (standard errors).
Significant differences were assessed using One-way ANOVA (Dunnett. Use Ci-Ctrl as control category for CiA ~CiH and CiP; use CiP for CiAP ~ CiHP).
Ci-Ctrl: control, Ci with P-free NBRIP adding; CiA ~ CiH: PSB treatment, Ci with A ~ H suspensions inoculation; CiP: TCP treatment, Ci with TCP supply andP-free NBRIP adding; CiAP~CiHP: Combination treatment, Ci with A~H suspensions inoculation and TCP supply.
PSB, TCP, and PSB*TCP: Two-way ANOVA for the factors PSB inoculation, TCP supply, the interaction of PSB inoculation ×TCP supply.
***: p < 0.001, **: p < 0.01, *: p < 0.05, −: p ≧ 0.05.

85

Table 3.3.3b Changes of labile P, moderately labile P and HCl-P in Ci inoculation experiment (mg-P kg-1 soil)
grouplabile P

relative to Ci

moderately labile P

labile P Increase

HCl-P decrease

7.04
7.96
7.41
3.33
5.37

Ci-Ctrl

6.84

CiA
CiB
CiF
CiG
CiH

8.98
9.88
10.01
8.23
8.44

2.14
3.04
3.17
1.39
1.60

CiP

8.58

1.74

CiAP
CiBP
CiFP
CiGP
CiHP

11.70
10.63
10.77
9.22
10.01

4.86
3.79
3.93
2.38
3.17

5.13

relative to CiP
labile P Increase
HCl-P decrease
3.12
13.15
2.05
10.37
2.19
8.15
0.64
6.85
1.43
7.22

relative to Ci
moderately labile P
Increase

5.80
5.11
5.11
5.18
5.38

0.66
-0.02
-0.02
0.04
0.25

5.14

0.00

6.14
5.29
5.37
5.94
5.52

1.01
0.16
0.24
0.81
0.39

Ci-Ctrl control; CiA ~ CiH PSB treatment, Ci with A ~ H strains inoculating；CiP TCP treatment, Ci with TCP supply；CiAP ~ CiHP Combination treatment, Ci with
A~H strains inoculating and TCP supply；86

To further clarify how PSB inoculation affected the soil labile and M labile P, Pearson correlation were calculated.
Table 3.3.4 Pearson correlation between PSB inoculation and soil physicochemical indicators
Labile P
PSB
Labile P
M labile P
pH
Oxalic—M labile P
pH
Oxalicin La without TCP.471*-.624**—.805**————.797**

ACP

Labile P—.831**.683**——.735**in La with TCP.536*—-.848**.803**-.527*-.471*
—
—
—

—
—
PSB
—
Labile P—
M labile P—
pH—
Oxalic**: p ＜ 0.01; *: p ＜ 0.05; -: p ≥ 0.05-*: Significant negative correlation; *: Significant positive correlation.621**

M labile P
pH
Oxalicin Ci without TCP——-.607**——-.559*-.476*————

in Ci with TCP—-.679**
—
—
—
—
—

ACP
—
—
—
—
.504*

.551*
—
—
—
—

As Table 3.3.4 showed that in both La with and without TCP, PSB inoculation showed a positive correlation with soil
M labile P, but no correlation showed with labile P. In in both Ci with and without TCP, PSB inoculation showed a87

positive correlation with soil labile P, while no correlation showed withsoil M labile P.
Considering the information above, in the PSB treatment of La,
strain B with a higher release capability for phytin caused the greatest
increase in soil labile P. It can be seen that in La, the three phosphataseactivities of the group inoculated with B were higher than those of thegroup inoculated with other strains, which may help B release certain
organic phosphates in the soil. At the same time, it can be seen that thedecrease in soil pH of the group inoculated with B was smaller than thatof the group inoculated with other strains, which is consistent with B's pHchanges in the phytin bottle. Overall, in La with low HCl-P content,
phosphatase may mainly be relied upon to change the soil P structure.
This can be seen in Table 3.3.4, where soil ACPase activity was positively
correlated with soil labile P content and M labile P content.
In the combination treatment of La, the supply of TCP has changed
the original P fractionation in the soil, thereby affecting the performanceof various strains in releasing P from the treated soil. Significant
decreases in soil pH were observed in the groups inoculated with strains
A and H, while the group inoculated with strain G exhibited higher acidphosphatase activity than the other groups. According to Table 3.3.4, soil
pH was negatively correlated with soil labile P content, and higher labile
P content was detected in the soil of group A with lower pH than in other88

groups. This may indicate that the pH decrease is one of the reasons forthe increase in labile P in the soil with high HCl-P. It is worth noting that,
although the soil pH in the group inoculated with strain H was the sameas that in group A, there was no significant increase in labile P comparedto other strains. Therefore, the pH decrease is not the absolute cause of
the increase in labile P in the soil with high HCl-P.
In PSB treatment of Ci, which was Ca-P rich soil, the largest pH
decrease was observed in the group inoculated with strain H, while themaximum acidic phosphatase activity was found in the group inoculatedwith G. However, the greater decrease in soil HCl-P and the larger
increase in labile P were observed in groups B and F. The correlation
analysis in Table 3.3.4 also indicates that soil pH and acidic phosphatase
are not related to labile P content in Ci with TCP.
In the combination treatments of Ci, the maximum pH decrease
occurred in the group inoculated with strain H, while the highest activityof acidic phosphatase was found in the group inoculated with strain B.
The maximum decrease in HCl-P and the maximum increase in labile P
were observed in the group inoculated with strain A. As shown in Table
3.3.4, similar to the Ci without TCP, the analysis results of Ci with TCP
also indicated that there was no correlation between soil pH and acidic
phosphatase with soil labile P.
It could be seen that PSB exhibited different P release efficiencies in89different environments. This might be related to the release strategies of
PSB itself (secretion of phosphatase or lowering of pH), as well as theinfluence of the environment. In this experiment, regardless of Ci or La,
strain B caused the maximum decrease in soil HCl-P when TCP was not
supplied. After TCP supply, strain A caused the maximum decrease in
HCl-P. Therefore, the same environmental changes might have different
effects on the phosphorus release efficiency of different PSB.

90

Chapter IV
PSB affect maize seedling P accumulation in sterilized soils(sterilized co-culture experiment)

To investigate whether PSB affects maize seedling P accumulation
in soils, a sterilized co-culture experiment was carried out. In thisexperiment, PSB strain A was inoculated into the roots of maize seedlings
growing in sterile La and Ci soils, with or without TCP added. Strain A
was selected because it caused an increase in labile P of 1.68 mg-P kg-1
soil in LaAP and 4.86 mg-P kg-1 soil in CiAP, which were higher thanother strains. One week later, strain A was found to have survived, andthere was no bacterial growth in the TCP treatment or the control. The
maize shoots and soils were then collected for measurement.

4.1 Materials and methods
4.1.1 Materials and treatments
Soils used in sterilized co-culture experiment are the same as 3.2.1,
and were set the treatments as follows (Table 4.1.1):

91

Table 4.1.1 Treatments and conditions
Soil and P
supplements

Treatments

Inoculation

Moisture support

Control

La / Ci 50g

2.5 mL P free NBRIP

12.5 mL sterile distilled water

PSB treatment

La / Ci 50g

2.5mL A suspension

12.5 mL sterile distilled water

TCP treatment

La / Ci 50g + 0.5g TCP 2.5 mL P free NBRIP

12.5 mL sterile distilled water

Combination
treatment

La / Ci 50g + 0.5g TCP 2.5mL A suspension

12.5 mL sterile distilled water

Add 5 mL P free Hoagland nutrient solution was added to support plant growth every other day.
Add sterile distilled water by weight.

Strain A suspension was prepared as thatin 2.1.2.1 (Shake flask culture).
Maize seed: Zea mays L. cv. Guidan 162;
Guangxi

ZhaoheSeed

Industry,

China(Fig.4.1.1).

Fig.4.1.1 Package
used maize seeds

4.1.2 Seedling cultureofThe flow of cultivation treatment was shown in Fig.4.1.2. Maize
seeds were well washed with tap water. After draining off the water, seeds
were washed once with 75% (v/v) ethanol. They were then rinsed off withsterile distilled water. The seeds were immersed in 1% (w/v) mercuric
chloride for 2 min and were rinsed with sterile distilled water. Sterilized
seeds were covered with moistened sterile paper towels and were placedin sterile petri dishes. Germination was conducted for 24 hr at 28°C.

92

Fig.4.1.2 Flow of the inoculation experiment

The germinated maize seeds were transplanted into culture
containers containing the control and treatment soils. To each container, 5
milliliters of P-free Hoagland nutrient solution (Hoagland and Arnon,
1950) and 12.5 milliliters of sterile distilled water were added. After a
24-hour incubation at 28°C, the maize seeds were carefully removed fromthe seedlings. Two point five milliliters of PSB suspension were added to
the PSB treatment and combination treatment, while 2.5 milliliters ofP-free NBRIP were added to the control and TCP treatment. Then, 5
milliliters of P-free Hoagland nutrient solution were added to eachcontainer every other day to support plant growth. Additionally, sterile
distilled water was added to maintain moisture, which was controlled by93

weight. Seven days later, the above-ground plant parts were harvested,
dried at 70°C, weighed, and measured to determine total P content. Zeropoint one gram of soil was used to confirm strain survival, while 1 gramof soil was air dried to measure soil phosphatase activity. The remaining
soils were lyophilized to measure P fractions and organic acid content.

4.1.3 Seedling P content measuring
The content of total P in plant shoot was determined by H2SO4
decomposition method:
Weigh 0.05g of each sample into a 25 mL glass test tube, and thenadd 1 mL concentrated H2SO4 into the tube. Place the test tube at 200 ℃
for heat preservation. After 5 minutes, add 200 μL 30% hydrogen
peroxide to the tube, and then add it every 7 minutes until the solution
becomes clear and transparent. After the solution becomes clear andtransparent, keep it at 200 ℃ for 2 hours to drive away the remaininghydrogen peroxide.
After cooling the solution, add distilled water to a constant volumeof 20 mL, and measure the P content in the solution with molybdenum
blue method. Then calculate the P content of the plant sample according
to the digested weight.

4.1.4 Soil P fraction, pH, organic acid and phosphatase measuring
94

Soil P fractions was measured by Hedley method which is the sameas 3.1.2. Soil pH was measured by pH meter, the content of organic acidin soil is detected by HPLC, and the enzyme activity is measured by kit,
which is the same as 3.1.3-3.1.5.

4.2 Results
4.2.1 Maize seedling P accumulation of two soils
Maize seedling shoot dry weight in La was generally heavier thanthat in Ci (Fig. 4.2.1a). PSB inoculation or TCP supply increased shoot
dry weight. Combination treatment showed heavier than single treatment
as PSB or TCP treatment. Relative to control, combination treatment
increased 74% in La and 51% in Ci.

Fig. 4.2.1a Dry weight of maize seedling shoot
Maize seedling shoot P content in La is almost half of that in Ci, andalmost same within La treatments (Fig. 4.2.1b).

95

Fig. 4.2.1b P content of maize seedling shootIn terms of P accumulation of maize seedling (Fig. 4.2.1c), LaA andCiA were higher than La-Ctrl and Ci-Ctrl, respectively. LaP and CiP werealso higher than La-Ctrl and Ci-Ctrl, respectively, LaAP was higher than
La-Ctrl, LaA and LaP, and CiAP was higher than Ci-Ctrl and CiP. There
were no significant differences between LaA and LaP and between CiA
and CiP.

Fig. 4.2.1c P accumulation of maize seedling shoot. Different letters
indicate significant differences (p < 0.05; one-way ANOVA, Tukey, n =3). Error bars = SE.

The results in Table 4.2.1 indicated that In La, PSB inoculation
increased the P accumulation by 0.082 mg, increase rate was around 31%;
TCP supply increased the P accumulation by 0.097 mg, increase rate was

96around 36%; and the combination treatment increased the P accumulation
by 0.217 mg, increase rate was around 81%. The absolute increase andincrease rate of LaAP was higher than that of LaA. In Ci, PSB inoculation
increased the P accumulation by 0.244 mg, increase rate was around 42%;
TCP supply increased the P accumulation by 0.200 mg, increase rate wasaround 35%; and the combination treatment increased the P accumulation
by 0.265 mg, increase rate was around 46%. The absolute increase andincrease rate of CiAP was similar to CiA.
In both soils, all treatments increased maize seedling P accumulation
higher than the control, and there was no significant difference between
the PSB treatment and the TCP treatment.
In terms of the increase in quantity, the combined treatment of LaAP
showed a greater increase compared to the combined increase of the
PSB-treated LaA and TCP-treated LaP, while it was lower in Ci soils.
In terms of the increase rate, PSB inoculation was more efficient in
Ci soil compared to La soil, while TCP supply showed a similar increasein both soils. However, the combined utilization of PSB and TCP in La
soil showed an increase nearly twice as much as in Ci soil.
In general, for La soil, although the overall P accumulation is low,
the combined utilization of PSB and TCP significantly improved theplanting effects. In Ci soil, PSB inoculation alone showed promising
results.
97

Table 4.2.1 Maize seeding P accumulation and variationgroup

P accumulation(mg-P/ shoot)

La-Ctrl

0.268±0.011c

Increase (mg)Increase rate %LaA

0.350±0.005b

0.082

30.60

LaP

0.365±0.009b

0.097

36.19

LaAP

0.485±0.016a

0.217

80.97

relative to La-Ctrl

relative to Ci-Ctrl
Ci-Ctrl

0.580±0.024c

Increase (mg)Increase rate %CiA

0.824±0.009ab

0.244

42.07

CiP

0.780±0.016b

0.200

34.48

CiAP

0.845±0.007a

0.265

45.69

P accumulation represent the mean of three replicates ± SE (standard errors).
Values in a column not followed by a common letter indicate significant differences (p < 0.05; one-way ANOVA, Tukey).
La, Ci control; LaA, CiA PSB treatment；LaP, CiP TCP treatment；
LaPA, CiPA Combination treatment.

4.2.2 Soils pH
In sterilized co-cultivation experiments, soil pH measurements wereconducted on different treatments, and it was found that inoculation with
PSB strain A did not significantly alter the soil pH values (Fig.4.2.2). In
La, the addition of TCP increased soil pH, while in Ci, the addition of
TCP had no significant effect on soil pH. When compared with the soil
inoculation experiments, it was found that after strain A was inoculated
into La, it caused a 10.3% decrease in soil pH, while in La planted with98

corn, strain A caused a 3.5% decrease in soil pH. Using LaP as a control,
strain A’s inoculation caused an 11.6% decrease in LaP soil pH, while in
LaP planted with maize seedling, there was a 1.4% increase in pH causedby strain A inoculation. In Ci, regardless of whether TCP was added ornot, strain A’s inoculation caused a slight decrease in soil pH, while in Ci
planted with maize seedling, strain A’s inoculation did not cause adecrease in soil pH. Thus, the alteration of soil pH by PSB is influenced
by the roots of plants to a certain extent.

Fig. 4.2.2 Soil pH of strain A inoculation experiment. The percentages
indicated on LaA, LaP, and LaAP represent the percentage of pH change
relative to La-Ctrl in each treatment; The percentages indicated on CiA,CiP, and CiAP represent the percentage of pH change relative to Ci-Ctrl
in each treatment.
4.2.3 Soils organic acid
Soil organic acids, mainly oxalic acid, were detected in alltreatments of sterilized co-culture of both soils (Fig.4.2.3ab). In La, theinoculation of strain A caused a 2.1% decrease in soil oxalic acid content,
while in TCP-added La, the inoculation of strain A also caused a 15.6%
decrease in soil oxalic acid content. In Ci, the inoculation of strain A
99

caused a 2.2% decrease in soil oxalic acid content, but in TCP-added La,
the inoculation of strain A did not cause a decrease in soil oxalic acidcontent.

Fig. 4.2.3a Content of organic acid in the sterilized co-culture soil

Comparing these results with the organic acid detection results of thesoil inoculation experiment with strain A, it was found that the soil oxalic
acid content in La and Ci, where no maize seedlings were planted, waslower than that in the soil where maize seedlings were planted. In termsof inoculation experiment, the inoculation of strain A caused 7.3% and85.9% decrease in La and TCP-added La's oxalic acid content, whichwere both greater than the decreases in soil oxalic acid content in thesame treatment where maize seedlings were planted. The inoculation ofstrain A also caused 71.4% and 60.0% decrease in Ci and TCP-added Ci's
oxalic acid content, which were both greater than the decrease in soil
oxalic acid content in the same treatment where maize seedlings wereplanted.

100

Fig. 4.2.3b Soil organic acid (mg kg-1 soil) of strain A inoculation
experiment. The value "0" represents trace amounts. The terms used in
the context are as follows: "Succinic" refers to succinic acid, "Citric"
refers to citric acid, "Maleic" refers to maleic acid, "Acetic" refers toacetic acid, "Malonate" refers to malonic acid, "Malic" refers to malic
acid, and "Oxalic" refers to oxalic acid.
It is worth noting that in the soil inoculation experiment with strain
A, a certain amount of malic acid was present in Ci and TCP-added Ci
where strain A was not inoculated, but it was not found after inoculation,
but it was not detected after inoculation. However, in the sterilized
co-culture experiment, malic acid was not detected in any of thetreatments of Ci, but a certain amount of malic acid was detected in alltreatments of La, except for the high content of oxalic acid.
The author considered that the decrease in soil oxalic acid content inboth experiments might be due to the consumption by the inoculated PSB.
However, in the soil where maize seedlings were planted, the root system101

might provide oxalic acid, which resulted in a smaller decrease in oxalic
acid content than in the soil where no maize seedlings were planted. As
for malic acid, it might also be consumed by PSB, but in the sterilized
co-culture experiment, maize roots in La might secrete more malic acidthan in Ci, resulting in a higher detection of malic acid in La where maize
seedlings were planted than in Ci where maize seedlings were planted (nomalic acid was detected in any treatments of Ci where maize seedlings
were planted).

4.2.4 Soils phosphatase
As shown in Figure 4.2.4a, the activities of soil acid phosphatase(ACPase) in the La treatments were lower than those in the Ci treatments.
In both soils, whether with or without added TCP, inoculation with PSB
strain A increased the activity of soil ACPase. Comparing the soil
inoculation experiment with strain A (Figure 4.2.4a'), it can be observedthat the presence of maize seedling roots reduced the soil ACPaseactivities in the La treatments, but increased them in the Ci treatments.

Fig. 4.2.4a ACPase activity of sterilized co-culture soil
102

Fig 4.2.4a' ACPase activity of strain A inoculation experimental soil.
The percentages indicated on LaA, LaP, and LaAP represent thepercentage of activity change relative to La-Ctrl in each treatment; Thepercentages indicated on CiA, CiP, and CiAP represent the percentage ofactivity change relative to Ci-Ctrl in each treatment.
The activity of neutral phosphatase (NPase) detected in the soil inthis study was approximately only 1% of the activity of acid phosphatase(ACPase) (Figure 4.2.4b). Similar to ACPase activity, the NPase activities
in the La treatments were lower than those in the Ci treatments, but thedifferences were smaller compared to the differences in ACPase activities
between the two soils. Inoculation with the PSB strain A also led toincreases in soil NPase activities. Compared to the unplanted soil (Figure
4.2.4b'), the presence of maize seedling roots had little influence on thesoil NPase activity in the La treatments. However, it increased NPaseactivity in the Ci treatments by approximately 30%, which was lower
than the increases in ACPase activities (approximately 50%-80%).

103

Fig. 4.2.4b NPase activity of sterilized co-culture soil

Fig 4.2.4b' NPase activity of strain A inoculation experimental soil.
The percentages indicated on LaA, LaP, and LaAP represent thepercentage of activity change relative to La-Ctrl in each treatment; Thepercentages indicated on CiA, CiP, and CiAP represent the percentage ofactivity change relative to Ci-Ctrl in each treatment.
Although the activity of alkaline phosphatase (AKPase) in the soil
was very low (less than 1% of ACPase), a positive effect of PSB on the
AKPase activity in various treatments can still be observed from Figure
4.2.4c: regardless of the addition of TCP, the inoculation of strain A
increased the AKPase activity in the corresponding soil treatments. The
AKPase activity in the Ci treatments remained higher than that in the La
treatments. Compared to the unplanted soil (Figure 4.2.4c'), the presenceof maize seedling roots had little influence on the soil AKPase activity inthe La treatments, but it led to an increase in AKPase activity in the Ci
104

treatments by approximately 40-60%.

Fig. 4.2.4c AKPase activity of sterilized co-culture soil

Fig. 4.2.4c' AKPase activity of strain A inoculation experimental soil.
The percentages indicated on LaA, LaP, and LaAP represent thepercentage of activity change relative to La-Ctrl in each treatment; Thepercentages indicated on CiA, CiP, and CiAP represent the percentage ofactivity change relative to Ci-Ctrl in each treatment.
In summary, in both the La and Ci treatments with maize seedlings,
inoculation with PSB strain A still increased the soil phosphatase activity.
Unlike in unplanted soil, the extent of increase is influenced not only bysoil type and the addition of TCP but also by the presence of plant rootsystems.

4.2.5 Soils fractions
As Table 4.2.5.1 showed, in sterilized co-culture experiment, La105

treated with PSB isolate A (LaA) showed differences in all fractions
except NaHCO3-Po relative to the uninoculated control soil (La-Ctrl),
while LaAP (relative to LaP) significantly changed all fractions except
HCl-P; CiA (relative to Ci-Ctrl) significantly changed all fractions except
NaOH-Po, while CiAP (relative to CiP) significantly changed all
fractions except NaOH-Po and HCl-P. TCP supply significantly increased
all except NaOH-Po fraction in LaP whereas it increased all except
NaOH-Pi and NaOH-Po in CiP. The interaction between PSB inoculation
and TCP supply was found to be significant for resin P, NaHCO3-Pi, and
NaOH-Pi in La, whereas for labile P (resin P, NaHCO3-Pi and
NaHCO3-Po) in Ci.
Observing the changes in labile P and M labile P in the soil, as
shown in Table 4.2.5.2, it was found that in La, all three treatments (PSB
treatment, TCP treatment, and combination treatment) showed an increasein M labile P higher than labile P. However, in Ci, all three treatments
showed an increase in labile P higher than M labile P. Comparing with the
inoculation experiment of strain A (Table 4.2.5.2'), it can be seen thatregardless of whether maize seedlings were planted in the two soils or not,the trend of changes in labile P and M labile P under the three treatments
was consistent. The difference is that in La with maize seedlings planted,
the soil M labile P content is slightly higher than that in unplanted La,
while in Ci with maize seedlings planted, both the soil labile P and M
106

labile P contents are slightly higher than those in unplanted Ci.
Additionally, the presence of roots also slightly increased the incrementof labile P and M labile P in each treatment of La and increased theincrement of labile P in each treatment of Ci. Therefore, the authors
considered that changes in soil P fractionation due to the presence ofroots might also have some degree of influence107

Table 4.2.5.1 Soil P fractions (mg-P kg-1 soil) in sterilized co-cultured La and Ci
GroupResin PNaHCO3-Pi

NaHCO3-Po

NaOH-Pi

NaOH-Po

HCl-P

Total

La-Ctrl
LaA
LaP
LaAP
PSB
TCP
TCP * PSB

0.027±0.003a
0.061±0.003b
0.564±0.006c
1.174±0.015d
***
***
***

0.023±0.002a
0.068±0.004b
0.361±0.006c
0.649±0.021d
***
***
***

0.212±0.004a
0.226±0.004a
0.256±0.006b
0.292±0.011c
***
***
-

14.795±0.265a
18.004±0.231b
40.991±0.341c
46.998±0.243d

9.431±0.230a
10.991±0.203b
10.113±0.234a
12.103±0.305c

585±16a584±10a2113±9b2112±12b

***
***
***

***
***
-

0.065±0.006a
0.007±0.002b
436±3c427±2c*****Ci-Ctrl
CiA
CiP
CiAP
PSB
TCP
TCP * PSB

1.86±0.02a
2.47±0.03b
3.89±0.09c
4.20±0.07d
***
***
*

3.82±0.08a
5.11±0.03b6.09±0.03c
6.92±0.08d
***
***
***

1.72±0.05a
2.19±0.05b
2.10±0.05b
3.32±0.05c
***
***
***

2.05±0.01a
2.39±0.01b
2.06±0.04a
2.40±0.02b***-

4.36±0.02a
4.39±0.02ab
4.30±0.09ab
4.49±0.04b*-

306±4a291±2b1704±8c1701±9c***-684±14a683±14a2170±12b2168±11b

***
-

***
-

Values represent the mean of three replicates ± SE (standard errors).
Significant differences in a column under each group are indicated by different letters (P≤0.05). Significant was analyzed with One-way ANOVA (Games-Howell).
La-Ctrl, Ci-Ctrl control; LaA, CiA PSB treatment；LaP, CiP TCP treatment；LaAP, CiAP Combination treatment；
PSB/TCP/PSB*TCP Two-way Anova for the factors PSB inoculation/TCP supply/ the interaction of PSB inoculation ×TCP supply.
***: p < 0.001, **: p < 0.01, *: p < 0.05, −: p ≧ 0.05.

108

Table 4.2.5.2 Changes of labile P, moderately labile P and HCl-P in sterilized
co-culture experiment (mg-P kg-1 soil)
grouplabile P
Increasemoderately labile P
Increase
La-Ctrl
0.26
relative to La-Ctrl
24.23
relative to La-Ctrl
LaA
0.36
0.10
29.00
4.77
LaP
1.18
0.92
51.10
26.87
LaAP
2.11
1.85
59.10
34.87
Ci-Ctrl
CiA
CiP
CiAP

7.40
9.77
12.08
14.44

relative to Ci-Ctrl
2.37
4.68
7.04

6.41
6.78
6.36
6.88

relative to Ci-Ctrl
0.37
-0.05
0.47

La-Ctrl, Ci-Ctrl control; LaA, CiA PSB treatment；LaP, CiP TCP treatment；LaAP, CiAP combination treatment；

Table 4.2.5.2’ Changes of labile P, moderately labile P and HCl-P in inoculation
experiment (mg-P kg-1 soil)
grouplabile P
Increasemoderately labile P
Increase
La-Ctrl
0.27
relative to La-Ctrl
23.71
relative to La-Ctrl
LaA
0.31
0.05
25.39
1.69
LaP
0.94
0.67
43.43
19.72
LaAP
1.94
1.68
53.78
30.07
Ci-Ctrl
CiA
CiP
CiAP

6.84
8.98
8.58
11.70

relative to Ci-Ctrl
2.14
1.74
4.86

5.13
5.80
5.14
6.14

relative to Ci-Ctrl
0.66
0.00
1.01

La-Ctrl, Ci-Ctrl control; LaA, CiA PSB treatment；LaP, CiP TCP treatment；LaAP, CiAP combination treatment；

4.3 Discussion
4.3.1 PSB inoculation affect maize seedling P accumulation
Based on the Pearson correlation analysis investigating therelationship between maize seedling P accumulation and PSB
inoculation, incorporating the measured soil physicochemical properties
mentioned above, the following conclusions can be drawn. In Ci
condition,
109

Table 4.3.1 Pearson correlation between PSB inoculation, maize P accumulation and soil indicators
P accum.

labile P

M labile P

pH

OxalicACPaseP accum.

in La (La-Ctrl & PSB treat.)
PSB
P accum.
labile P
M labile P
pH

-

.857*
-

.878*
.859*

-.919**-.818*

-

-

.842*

-

-

M labile P

pH

OxalicACPasein Ci (Ci-Ctrl & PSB treat.)

OxalicPSB
P accum.
labile P
M labile PpHlabile P

in La with TCP (TCP treat. & PSB treat.).935**.942**-.939**.938**-

Oxalic.828*-.866***: p ＜ 0.01; *: p ＜ 0.05; -: p ≥ 0.05-*: Significant negative correlation; *: Significant positive correlation

110

.866*
.956**.947**---.958**.812*.924**--

in Ci with TCP (TCP treat. & PSB treat.)-.996**.984**.828*-

maize seedling P accumulation is positively correlated with PSB
inoculation. In the La condition, regardless of the addition of TCP, nodirect relationship between PSB inoculation and maize seedling P
accumulation was found. However, observations revealed that in the La
condition, maize seedling P accumulation is negatively correlated withsoil pH, soil pH is negatively correlated with soil M labile P, and soil M
labile P is positively correlated with PSB inoculation. These relationships
can be expressed using the following proportional relationships (Note:"∝" indicates a positive correlation, not proportionality):
P accum. ∝ - pH ∝ - M labile P ∝ PSB
In the La with TCP, maize seedling P accumulation is negatively
correlated with soil oxalate content, soil oxalate content is negatively
correlated with soil acid phosphatase activity, and soil acid phosphataseactivity is positively correlated with PSB inoculation. These relationships
can be expressed using the following proportional relationships:
P accum. ∝ - Oxalic ∝ - ACPase ∝ PSB
Therefore, although maize seedling P accumulation is not directly
related to PSB inoculation in the La condition, an indirect positive
correlation can still be established between the two through soil
physicochemical properties.
Additionally, in the Ci condition with TCP, no direct or indirect
relationship was found between maize seedling P accumulation and PSB
111

inoculation. From Table 4.2.1, it can be seen that there is no significant
difference in maize seedling P accumulation between PSB inoculation
alone and combined inoculation. However, TCP supply has a significant
impact on maize phosphorus accumulation. Therefore, in the combined
treatment, it is difficult to determine whether it is the PSB inoculation or
TCP supply that is responsible for the effect.
Considering the release of soil P by PSB inoculation, it can beobserved that PSB inoculation positively affects soil labile P content in
the La, La with TCP, and Ci conditions, effectively facilitating the releaseof soil P.

4.3.2 Exploration of Factors Influencing Maize Seedling P Accumulation
In order to investigate the impact of PSB inoculation on maize
seedling P accumulation in the sterilized co-culture experiment, theauthor employed multiple linear regression analysis to analyze therelevant variables.
To establish a multiple linear regression model with [maize seedlingP accumulation] as the dependent variable and [soil type], [TCP supply],and [PSB inoculation] as independent variables, we can set thecontinuous numerical variable of [maize seedling P accumulation] as thedependent variable and the categorical variables of [soil type], [TCP
supply], and [PSB inoculation] as independent variables. We can assign112

La = 0 and Ci = 1 for [soil type], no supply = 0 and supply = 1 for [TCP
supply], and no inoculation = 0 and inoculation = 1 for [PSB inoculation].

Table 4.3.2a Model Summary
Model

R

R Square

1

.940

a

Adjusted R Square.884

Std. Error of the Estimate.867

Durbin-Watson.08433045

2.128

a. Predictors: (Constant), TCP supply, PSB inoculation, Soil type
Dependent Variable:maize seedling P accumulation

Fig.4.3.2 Normal P-P plot of regression standardized residual dependent
variable: maize seedling P accumulation
Table 4.3.2b Coefficients
Unstandardized Coefficients

Standardized Coefficients

Model

B

1

(Constant)

.247

.034

Soil type

.390

.034

.862

PSB inoculation

.128

.034

.282

TCP supply

.113

.034

.250

Dependent Variable:

Std. Error

Beta

Collinearity Statistics
Tolerance

VIF

.000

1.000

1.000

.001

1.000

1.000

.004

1.000

1.000

p
.000

maize seedling P accumulation

113

Based on the findings from Table 4.3.2a, the R Square statistic valueof 0.884 indicates that the three independent variables in the present study,
namely [soil type], [PSB inoculation], and [TCP supply], can explain
approximately 88.4% of the variation in the dependent variable, maize
seedling P accumulation. Additionally, as observed in Fig. 4.3.2, theregression standardized residuals exhibit a reasonably normal distribution.
Moreover, referring to the results in Table 4.3.2b, the collinearity
statistics indicate a lack of multicollinearity among the independent
variables, as evidenced by the VIF values of 1. This implies that theexamined variables do not substantially influence each other. Further
analysis from Table 4.3.2b reveals that all three independent variables
exhibit significance levels (p) below 0.05, indicating their statisticallysignificant effects on the dependent variable. Therefore, the current model
exhibits a good fit.
According to the regression results, the Unstandardized Coefficients(B values) for [soil type], [PSB inoculation], and [TCP supply] areestimated to be 0.39, 0.128, and 0.113, respectively. These values suggest
that, compared to planting in La, cultivating maize in Ci soil type leads toan increase of 0.39 mg-P shoot-1 in P accumulation. Similarly, PSB
inoculation enhances maize seedling P accumulation by 0.128 mg-P
shoot-1 compared to non-inoculation, while TCP supply contributes to arise of 0.113 mg-P shoot-1 compared to no supply. Based on these114regression findings, the following prediction equation can be derived:1) Maize seedling P accumulation= 0.247 + (0.39×soil type) + (0.128×PSB inoculation) + (0.113×TCP supply)

Using the prediction equation, the maize seedling P accumulation in
the co-cultivation experiment was as follows:

Table 4.3.3a Predicted maize seedling P accumulation (mg-P shoot-1) based on the
regression equation
Control
PSB treatment
TCP treatment
Combination treatment
La
0.247
0.375
0.360
0.488
Ci
0.637
0.765
0.750
0.878

Table 4.3.3b Actual measured maize seedling P accumulation (mg-P shoot-1)
Control
PSB treatment
TCP treatment
Combination treatment
La
0.268
0.350
0.365
0.485
Ci
0.580
0.824
0.780
0.845

As observed, the predicted values closely approximate the actual
measured values, indicating that the equation provides a good fit.
Based on the co-culture experiments conducted separately with La
and Ci soil types, an analysis of the factors influencing maize seedling P
accumulation was performed. Keeping [maize seedling P accumulation]
as the dependent variable, the independent variable [soil type] wasremoved, while the independent variables [PSB inoculation] and [TCP
supply] were retained. Regression analysis was conducted. Model115

diagnostics revealed the absence of multicollinearity among theindependent variables, and the regression standardized residuals exhibiteda roughly normal distribution. Based on the model, the prediction
equation is written as follows:2) Maize seedling P accumulation in La= 0.258 + (0.101×PSB inoculation) + (0.116×TCP supply) R²=0.601
3) Maize seedling P accumulation in Ci= 0.625 + (0.154×PSB inoculation) +(0.110×TCP supply) R²=0.531
From the three regression results, when conducting a regression
analysis of factors influencing maize seedling P accumulation using theco-cultivation results of two soil types, all three independent variables
have a significant impact on maize seedling P accumulation. Among them,[soil type] has the greatest influence, followed by [PSB inoculation], and[TCP supply] has the smallest influence. When conducting a regression
analysis using the co-cultivation results of La, it was found that both
[PSB inoculation] and [TCP supply] have a significant impact on maize
seedling P accumulation, with the impact of [TCP supply] slightly higher
than that of [PSB inoculation]. When conducting a regression analysis
using the co-cultivation results of Ci, it was found that both [PSB
inoculation] and [TCP supply] also have a significant impact on maize
seedling P accumulation, with the impact of [PSB inoculation] slightly
higher than that of [TCP supply]. In conclusion, the magnitude of PSB's
116

impact on maize seedling P accumulation varies depending on the soil
environment and different treatments, but the impact of PSB is positiveand certain.

117Chapter V

PSB affect maize seedling P accumulation in natural soil
(non-sterilized co-culture experiment)

In this chapter, a non-sterilized co-culture experiment was conducted
to investigate the effect of PSB on the rhizosphere bacterial community,
using fresh La as the cultivation substrate. Non-fresh soil (Shanxi
Cinnamon soils) was not utilized in this step due to its incomplete
microbial structure. Maize seedlings were transplanted on the first day
after sprouting, and the soil was collected and potted on the same day.
For soil DNA extraction, rhizosphere soil samples were collected on
the day of collection. Leveraging long-read sequencing technologies,
these samples were subjected to single-molecule real-time (SMRT)
sequencing on PacBio platforms, which generated circular consensus
sequences (CCS). The CCS reads were then filtered, clustered, andde-noised to generate full-length amplicon tags for species annotation andabundance analysis. Further analyses included alpha diversity, beta
diversity, differential analysis between groups, correlation analysis,
function prediction, and more.

5.1 Materials and methods
5.1.1 Materials and treatments
118

Strains A, B, F, G, and H were utilized for non-sterilized co-culture.
However, during the culture process, groups B and H experienced
non-experimental fatalities. Consequently, the results only reflect theoutcomes of groups A, F, G in the PSB treatment, control, and the P
treatments.
The PSB suspension was prepared as described in section 2.1.3(Shake flask culture).
The inoculation experiments consisted of two treatments (Ptreatment and PSB treatment) and one control (Table 5.1.1). All
treatments and the control employed the same soil, supplemented with
TCP.

119

For the PSB treatment, 25 mL of PSB suspension was inoculated.
For the P treatment and control, 25 mL of P-free NBRIP solution wasused. Subsequently, 125 mL of sterilized distilled water was added toeach container to maintain moisture. In addition, 5 mL of P-free
Hoagland nutrient solution was added to both the control and PSB
treatment, while a complete nutrient solution was provided to the P
treatment every other day to support plant growth. The treatments andcontrol were cultured at 28°C for 30 days.

Table 5.1.1 Treatments and conditions of the experiment

Treatments

Soils and P
supplements

Inoculation

Moisture support

HoaglandControl

La 500g + 5g TCP 25 mL P free NBRIP

125 mL sterile distilled water

P freeP treatment

La 500g + 5g TCP 25 mL P free NBRIP

125 mL sterile distilled waterComplete nutrition

PSB treatment La 500g + 5g TCP 25mL PSB suspensions 125 mL sterile distilled water
Add 5 mL Hoagland nutrient solution was added to support plant growth every other day.
Add sterile distill water by weight.
28℃, 30 days.

120

P free

The scheme of soil preparation was shown in Fig.5.1.1.1.

The soilsamples were collected from an outdoor field and subsequently cleared ofstones and other non-soil materials to the greatest extent possible. The
soil was then passed through a 2 mm sieve. To ensure uniformity, the soil
was evenly spread on sterile plastic cloth. The soil samples were divided
into 21 large grids, with each large grid further divided into 21 small
grids. One small grid soil sample was taken from each large grid andcombined to create a composite sample. In total, 21 composite soil
samples were obtained.
For each composite sample, 500 g of soil was weighed and mixed
with 5 g of TCP. The mixture was thoroughly blended and transferred toindividual pots. All equipment used in the process was disinfected with
75% alcohol to prevent the introduction of non-soil bacteria.

121Fig.5.1.1.1 Scheme of soil preparation in this experiment

Flow of the cultivation experiment was shown in Fig.5.1.1.2. The
seed sterilization, germination, transplant, and culture followed theprocedure outlined in section 4.1.2. After one month, both the plants andthe rhizosphere soil were sampled.

Fig.5.1.1.2. Flow of cultivation experiment

The aboveground portion of the plants was harvested and weighed,and various indicators such as height and leaf width were measured.
Following the measurements, the plants were dried to a constant weightin a constant temperature oven set at 70 ℃. Subsequently, the dried plants
were pulverized for the measurement of total P.

122

To collect rhizosphere soil, carefully extract the maize roots from thecultivation pot, gently shaking them to remove bulk soil. Place a sterile
bag over the maize roots after removing thebulk soil, shake the roots, causing the soil
tightly attached to the roots to fall into thesterile bag, thereby collecting rhizospheresoil (Fig.5.1.1.3).
Fig. 5.1.1.3 Collection of the rhizospheresoil in a bag
The collected rhizosphere soil was immediately weighed and used
for soil DNA extraction. One gram of soil was air-dried to measure soil
phosphatase activity, while the remaining soil samples were lyophilized
to measure organic acid content.

5.1.2 Soil pH, organic acid and phosphatase measuring
Soil pH was measured by pH meter, the content of organic acid insoil is detected by HPLC, and the enzyme activity is measured by kit,
which is the same as 3.1.3-3.1.5.

5.1.3 Seedling P content measuring
The content of total P in plant shoot was determined by H2SO4
decomposition method as same as 4.1.3.
123

5.1.4 Soil DNA extraction
TGuide S96 Magnetic Soil DNA Kit (Tianggen Biotech) was used to
extract soil DNA.

5.1.5 Amplicon sequencing
Soil DNA was sequenced by Biomarker Technologies Co., Ltd.,
Beijing, China.

5.1.6 Bioinformatic Analysis
Bioinformatic Analysis was conducted through BMK Cloud(Biomarker Technologies).

5.2 Results
5.2.1 Soil physicochemical indicators
Regarding pH, none of the treatments significantly altered soil pH or,in other words, the changes were almost negligible (Table 5.2.1). In
Chapter 4, the combination treatment of LaAP and the TCP treatment of
LaP did not result in any significant changes in soil pH. The results wereconsistent between the two.
In terms of soil phosphatase activity, the PSB treatment slightly
increased the activity, while the soluble P treatment reduced the activity
124

(Table 5.2.1). Similar trends were observed for neutral phosphatase.
The soil oxalic acid content showed a significant decrease in the
PSB treatment, especially in groups A and F (Table 5.2.1). The soluble P
supply only caused a minor decrease. A similar pattern was observed fortartaric acid, while malic acid showed a significant decrease in group A
and P treatment but an increase in groups F and G. Tartaric acid was
hardly detected in both Chapters 3 and 4, although it was present at
considerable levels in the experimental soil. This could be due to thedecomposition of tartaric acid after autoclaving of the soil.

125

Overall, in terms of physicochemical indicators, PSB inoculation had a significant impact on the content of organic
acids in the soil, particularly oxalic acid content, and also had some influence on phosphatase activity.

Table 5.2.2 Soil physicochemical indicators
ACPaseNPase

AKPaseOxalicTartaric

Malic

Treatment
and
Group

pH

Control

7.20

1.19

4.79

2.72

794.80

84.70

33.40

P

7.20

1.01

3.31

2.34

623.30

79.30

16.20

A

7.23

1.52

5.74

2.81

297.90

19.80

18.90

F

7.20

1.38

5.22

2.62

291.80

38.10

42.60

G

7.19

1.66

6.31

3.09

521.20

47.00

54.50

×106 U/g soil——×10³ U/g soil——126——————mg/kg soil——————

5.2.2 Maize seedling physiological indicators
From the physiological indicators of maize, P treatment increased
the indicators significantly while PSB treatment did not (Table 5.2.2).

Table 5.2.2 Physiological indicators of maize seedlings
Leaf

Fresh

Dry

Shoot P

Shoot P

Heightwidth

weightweightcontent

accumulation

Treatment

cm

cm

g

g

mg-P/g DW

mg

Control

62.67

2.93

18.64

1.88

4.96±0.05

9.34±0.78

A

66.00

3.43

23.3

2.28

5.2±0.07

11.63±0.80

F

65.00

3.37

22.71

2.17

4.92±0.18

10.54±0.72

G

62.33

3.6

22.24

2.16

5.22±0.15

11.08±0.54

P

89.00

4.33

40.01

3.74

7.37±0.20*

28.71±0.47*

Fig.5.2.2 Appearance of maize plants at harvest

It can be observed that inoculating PSB in a 7-day sterilized
co-culture of 2.3 significantly increased P accumulation in maize
seedlings. However, in non-sterilized co-culture, although the growth of127

maize seedlings with PSB inoculation appeared better than that of thecontrol group, the difference was not statistically significant (Fig.5.2.2).
This can be attributed to two factors: Firstly, the duration of the culture in
the two experiments differed. Secondly, PSB colonization and survival
were influenced by other soil microorganisms in the non-sterilized
co-culture. Hence, there are likely variations in the colonization orsurvival effects of PSB and the efficiency of P release into the soil
between the two co-culture experiments.

5.2.3 Rhizosphere bacteria species annotation and taxonomic
The following number of species were annotated in this sequencing：
Table 5.2.3 Statistics of species annotation
SamplecontrolA
F
G
P

Phylum
22
21
21
23
23

Class
42
40
43
45
45

Order
89
85
95
99
96

Family
143
126
152
153
151

Genus
220
199
252
247
245

Species
321
281
375
376
369

It can be seen from Table 5.2.3 that the species annotated in the treatment
inoculated with A were less than other treatments, and other inoculationand soluble P supply slightly increased the species in the soil.
Mapping comparison of the top 30 genera among groups shows thatA

has

relativelyhighabundanceon

Lactiplanatibaillus

andStaphylococcus, and the highest relative abundance of control is
128Candidatus Udaeobacter and Paenibacillus, the relative abundance of G
on Flavoisolibacter is higher than that of other treatments. In the P
treatment with sufficient P supply, the relative abundance ofHaliscomenobacter is higher (Fig. 5.2.3.1).

Fig. 5.2.3.1 Histogram of top 30 species distribution at genus level

It can be observed that in the P treatment and PSB treatment, thethree replicates of groups F and G exhibit consistent high-abundance
species, and the differences among the treatments or groups are also
evident. The three replicates of the Control treatment also share somesimilar high-abundance species composition, but control2 shows
significantly higher abundance in these species compared to the other two
129replicates. In group A, apart from the three high-abundance genera, there
are hardly any other genera present in A2. The distribution ofhigh-abundance genera in A1 and A2 also differs significantly, indicatingpoor reproducibility (Fig. 5.2.3.2).

Fig. 5.2.3.2 Heat map of species richness clustering

1305.2.4 Rhizosphere bacterial diversity
Simpson index shows that PSB and the supplied soluble P did not
significantly alter species diversity; the Chao1 index shows that PSB andthe supplied soluble P did not significantly alter species richness(Fig.5.2.4.1).

Fig. 5.2.4.1 α diversity at genus level
β diversity, as assessed through Principal Component Analysis(PCoA), revealed significant differences among the treatments in the PC2
direction (Fig.5.2.4.2). This indicates that the treatment had an impact on
the composition of rhizobacterial communities.

131

Fig. 5.2.4.2 Analysis of similarities (Anosim) at genus level

Furthermore, Anosim was performed to assess inter-group andintra-group differences among the samples (Fig.5.2.4.3). The calculated R
value was 0.768, indicating that the inter-group difference in this sample
grouping was greater than the intra-group difference (p = 0.001). Thissuggests high test reliability. The figure depicts β distances greater than0.5, while intra-group β distances are less than 0.4.

132

Fig. 5.2.4.3 Analysis of similarities (Anosim) at genus level

5.2.5 Difference of rhizosphere bacteria between groups
Two pictures were selected from the Marker intergroup ambiguity
histogram, one of which showed that the marker species in treatment A,
Enterobacter, had a high abundance in only one sample (Fig.5.2.5a).
Strain A happened to belong to Enterobacter. The results of thisinvestigation may be due to sampling errors, or it may be due to the highdegree of colonization of A in this sample.

133

Fig. 5.2.5a Marker intergroup abundance histogram

In addition, it can be seen from the following figure that inoculationof PSB increases the abundance of uncultured bacteria (except A2)(Fig.5.2.5b).

Fig. 5.2.5b Marker intergroup abundance histogram1345.2.6 Correlation network of rhizosphere bacteria

Fig. 5.2.6 Correlation network of top 50 correlated genus
135

Spearman rank correlation analysis was used to calculate thesignificant correlation among the top 50 genera with the highest
abundance in each sample (Fig.5.2.6, Table 5.2.6). The results showedthat Lactiplanatibaillus and Staphylococcus, which had the highest
abundance in the sample of treatment A, showed negative correlation
with other genera with significant correlation. These two genera have thehighest abundance in treatment A, which is likely to result in that the
annotation species found in treatment A are less than those in othertreatments.

Table 5.2.6 Edge properties
SourceTargetweightLactiplantibacillus

Vicinamibacter
unclassified_Acidobacteriales
Acidipila_Silvibacterium
RB41negative
Gemmatimonas
Pseudomonas
SWB02
Nordella-0.6068-0.5779-0.5784-0.5644-0.5779-0.6536-0.5317-0.7246

Staphylococcus

unclassified_Acidobacteriales
unclassified_Gemmatimonadaceae
MND1
unclassified_env.OPS_17
SWB02
Pseudolabrys-0.6036-0.5754-0.5149-0.6052-0.5564-0.5260

5.2.7 Functional genes prediction
It can be observed that Group F shows a significant difference136compared to the control in terms of predatory exparasitic, with the mean

Fig.5.2.7 Functional genes prediction of the rhizosphere bacterial
communities

proportion of Group F being higher than that of the control. Group G is137

predicted to have significant differences compared to the control in
multiple functional aspects of nitrogen cycling, particularly excelling inhydrocarbon degradation compared to both the control and P treatment. P
treatment, compared to the control, exhibits advantages in certain genes
associated with carbon and nitrogen cycling. Additionally, Group F andGroup G are predicted to have higher proportions in ureolysis
functionality compared to P treatment. Moreover, the mean proportion ofGroup G in anoxygenic_photoautotrophy is lower than that of P treatment.
No significant differences in functional gene prediction were foundbetween Group A and the control, as well as between P treatment and thecontrol.

5.2.8 Canonical Correspondence Analysis of Environmental Factors andMaize Seedling P accumulation
The Canonical Correspondence Analysis (CCA) was conducted toassess the relationship between environmental factors, including PSB
inoculation, soluble P supply, and maize seedling P accumulation, withthe rhizobacterial community (Fig.5.2.8). As it revealed, Pseudomonas,
which genus strain G belong to, was showed a positive correlation withmaize

seedlingPaccumulation.

The

genus

Flavisolibacter,

Lactiplantibacillus and Staphylococcus which exhibited the highest
relative abundance in the groups F, G and A, were positively correlated138

with PSB inoculation. However, none of these bacterial genera, including
Paenibacillus (the dominant genus in the control), exhibited a significant
positive correlation with maize seedling P accumulation.

Flavisolibacter
Lactiplantibacillus
Staphylococcus

Fig. 5.2.8 Canonical Correspondence Analysis of Environmental Factors
and Maize Seedling P accumulation.

CCA1 and CCA2 show low explanatory power,suggesting weakinflu-ence of the selected environmental factors on bacterial composition.
To further investigate the relationship between exogenous PSB andindigenous rhizobacterial communities, consider expanding environment139

al factors or increasing the sample size.

5.3 Discussion
In this chapter, three strains (A, F, and G) were employed toinoculate maize roots in order to investigate their effects on the maizerhizobacterial community. The results were then compared with
non-inoculated maize roots (control) and well-nourished maize roots (Ptreatment).
Based on the results of soil and maize seedling physiologicalindicators, it is evident that the changes in soil indicators were not as
pronounced as observed in Chapter 4. Additionally, the maize seedling
physiological indicators did not exhibit a significant increase in the PSB
treatment, suggesting that the PSB strains, especially strain A, did noteffectively enhance the planting effects compared to the soluble inorganic
P nutrient in this experiment.
To investigate the reason why the PSB strains did not exhibit similar
growth-promoting efficiency as observed in sterilized soil, an analysis of
the rhizobacterial community was conducted. The results of the species
annotation statistics revealed that the strains inoculated in the soil (A:Enterobacter chuandaensis; F: Klebsiella aerogenes; G: Pseudomonas
hunanensis) did not become dominant species in the rhizobacterial
community (they did not even rank among the top 30 abundant species).
140

Furthermore, the inoculation of exogenous PSB did not significantly alter
the bacterial abundance of maize roots (α Diversity), but it did affect thecommunity composition (β Diversity).
The results of Anosim also confirmed the effectiveness of sample
grouping, indicating that the inter-group difference was greater than theintra-group difference, and the sample grouping was statisticallysignificant.

Additionally,

Markerspecifications

betweengroupsdemonstrated that the inoculation of exogenous PSB increased therelative abundance of bacteria in uncultivated soil. Functional gene
prediction revealed that the hydrocarbon degradation capability in the Gtreatment was stronger compared to the control and P treatments.

141

Table 5.3 Pearson correlations within control and PSB treatment

PSB
Fresh weightFresh weight

Dry weightP accum.

pH

ACPaseOxalic

.676*

.603*

—

—

.707*

-.894**.961**.866**—.603*-.656*.955**—.630*-.591*—.749**———

Dry weightP accum.
pH
ACPase—142

Pearson correlation analysis was conducted to investigate therelationship between PSB inoculation and various indicators of maize andsoil (Table 5.3). The results revealed that PSB inoculation had a positive
influence on the fresh weight and dry weight of maize plants. It also
positively affected the soil acid phosphatase activity and exhibited anegative correlation with soil oxalic acid content. Although there was nodirect correlation observed between maize seedling phosphorus
accumulation and PSB inoculation, it was observed that maize seedling P
accumulation was positively correlated with soil acid phosphatase activity.
Therefore, it can be inferred that:P accum. ∝ ACPase ∝ PSB
In other words, PSB inoculation established an indirect positive
connection between soil acid phosphatase activity and maize seedling P
accumulation.
In general, based on the results of maize seedling physiologicalindicators, it can be observed that the PSB strains used in this study did
not improve maize yield as effectively as soluble inorganic P nutrients.
Additionally, strain A did not significantly enhance maize seedling P
accumulation as observed in the sterilized soil co-cultivation. As
mentioned earlier, the soil environment influences the release of soil
phosphorus by PSB, thereby affecting maize seedling P accumulation. In
this experiment, PSB strains should coexist or compete with the143indigenous microbial community in the niche, further influencing theirability to release soil phosphorus and promote maize seedling P
accumulation.
Through amplicon analysis, it was found that the strains (A, F, G)
inoculated into the soil did not become dominant bacteria, except for the
relatively higher abundance of strain A in A2 samples. Furthermore, while
PSB inoculation showed a positive correlation with the abundance ofdominant bacteria in groups A, F, and G, these dominant bacteria did notexhibit a positive correlation with maize seedling P accumulation.
However, if soluble P supply is not considered and only Pearson
correlation analysis is conducted among the three groups (control and
PSB treatments), it can be observed that PSB inoculation establishes anindirect positive correlation with maize seedling P accumulation throughsoil acid phosphatase activity. The potential reason for this discrepancy islikely due to the limited inclusion of environmental factors or a small
sample size (indicated by low CCA1 and CCA2 values). Further
investigation is needed to clarify and explore the relationship between
these factors more comprehensively and explicitly.

144

General Discussion

Chapter VI

6.1 PSB P release ability
In this research, PSB demonstrated high potential for dissolving TCP,
but they had weak effects on FePO4 and AlPO4, which aligns with thefindings of Chung et al. (2005) and Jiang et al. (2020). These results may
be attributed to the isolation method, where TCP was used as the P source
for the isolation medium. Furthermore, these PSB altered the P fractionsof Ci and La. Previous studies have shown that PSB isolates belonging to
the same genus exhibit different phosphorus release abilities in various
soils. For instance, Pseudomonas sp. in yellow cinnamon soil (Liu et al.,2019), Klebsiella sp. in soil from a subtropical moso bamboo forest (Liuet al., 2022), and Pantoea rodasii in sandy loam (Lee et al., 2016)enhanced the available P content. Additionally, Enterobacter sp.
reportedly increased P uptake in sugarcane, which is consistent with thefindings of strain A in this study, showing increased P accumulation inmaize (Safirzadeh et al., 2019).
The five PSB used in this research exhibited different abilities torelease phosphate sources (Figure 2.3.1), which may be attributed to their
distinct P release mechanisms (Wang et al., 2014; Zhu et al., 2009),
consequently leading to variations in their efficiency to release P from145

soils. Moreover, the efficiency of these PSB in releasing P from TCP in
shake flask cultures did not correspond to their efficiency in releasing
HCl-P in soils. For example, strains F and G released more abundant P
from TCP than the other three strains (Figure 2.3.1), but they did notcause a stronger decrease in sterilized soils compared to the other strains(Table 3.3.3b and 3.3.3b). One possible reason for this discrepancy is thatthe environment provided by the culture medium in shake flasks greatly
differs from that of the soil. It has been reported that the buffering
capacity of soils can limit the solubilization of soil phosphates by
microorganisms (Cabala-Rosand and Wild, 1982; Gyaneshwar et al.,1998), and PSB organic acid production can be influenced by different
nitrogen and carbon conditions in the soil (Cuningham and Kuiak, 1992).
Some studies have also indicated that PSB with high solubility to TCP in
the medium did not enhance P accumulation in plants (Poonguzhali et al.,2008; Collavino et al., 2010). Bashan et al. (2013) argued that TCP is nota suitable selection factor for PSB testing. From this research, we also
believe that evaluating the release ability of PSB to soil P solely based on
their release ability to TCP is inaccurate.
This study revealed that although different strains may cause varying
changes in soil P fractionation, the trend of changes in the same soil
remains consistent. For La, a soil with pH 5.0-5.5 and high PRP, its soil P
fractionation are predominantly composed of moderately labile P. With
146

the inoculation of PSB, regardless of TCP supply, La primarily increased
moderately labile P, with a small amount of released P contributing tolabile P. Delfim et al. (2020) also reported significant increases in
NaOH-Pi levels due to changes in the P pools of Andisol and Ultisol with
pH 5.5 and 5.8, induced by B. thuringiensis, which is consistent with ourfindings. For Ci at pH 8.0 with high HCl-P content, PSB primarily
increased labile P. Thus, the changes in soil P fractionation caused by
PSB can be considered soil-type dependent. Our research results suggestthat the varying phosphate release abilities of PSB result in differentchanges in soil P fractionation, but these changes do not significantly
alter the original soil P fractionation.
In the inoculation experiment, Pearson correlation analysis was
conducted for two soils. Both soils have four treatments: with TCP supply(control and PSB treatment) and without TCP supply (TCP andcombination treatments). No significant correlation was found between
labile P and HCl-P (data not shown). Additionally, it was observed thatwhen the content of calcium phosphate in the soil was high (combination
treatment of La, PSB, and combination treatments of Ci), the decreases in
HCl-P caused by the inoculation of these five PSB exceeded the increases
in labile P. A study by Liu et al. (2022) also demonstrated that inoculating
Klebsiella in soil from a subtropical moso bamboo forest (HCl-P
approximately 65 mg-P kg-1 soil) resulted in a greater decrease in HCl-P
147

compared to the sum of the increases in H2O-P, NaHCO3-Pi, and
NaHCO3-Po.

6.2 Maize seedling P accumulation
The effects of PSB on improving plant P uptake and promoting
biomass have been widely reported (Biswas et al., 2022; Dasila et al.,2022; Liu et al., 2022; Sabra et al., 2022). The results of our study also
indicated that PSB promoted the maize seedling P accumulation: themaize seedling P accumulation of the PSB treatment and the combination
treatment were greater than the control.
All treatments of the two soils were found to have significantly
increased maize seedling P accumulation, but the difference between the
PSB treatment and the TCP treatment was not significant. Maize seedlingP accumulation of all treatments in La was less than that in Ci. Regarding
the increase rate of maize seedling P accumulation, LaA was found to beless than CiA, and LaP was similar to CiP, but LaAP was much higher
than CiAP. Although P accumulation is low, it is apparent from theincrease rate that the combination effect for La is strong. For Ci, PSB
inoculation alone worked well, but TCP did not bring much benefit.
Findings from linear regression incorporating the soil type, PSB
inoculation, and TCP supply on maize seedling P accumulation indicatedthat the soil type, PSB inoculation, and TCP supply had significant
148

positive effects on maize seedling P accumulation, and soil type had thegreatest effect on maize seedling P accumulation. For La only, PSB
inoculation and TCP supply had significant positive effects on maize
seedling P accumulation, and TCP supply’s effect was slightly higher thanthat of PSB inoculation. But in Ci, the effect of PSB inoculation is greaterthan that of TCP supply. Although the soil type has a great influence on
plant P accumulation, its selectivity in agricultural production is minimal.
Therefore, PSB and P fertilizer are anticipated as effective means toincrease crop yield.

6.3 Maize rhizosphere bacteria community
Whether exogenous PSB have impact on indigenous microbialcommunities is the result of multiple factors. For example, thecompetition of exogenous strains for niche, that is, the colonization ofexogenous strains in the environment. The success of colonization is not
only related to the strain, but also to the soil environment and soil
microbial environment (Liu J. et al., 2020).
Wang et al. (2017) studied the colonization of Bacillus in therhizosphere of Chinese cabbage by using GRP marker method, and foundthat the population of Bacillus remained in a stable range after 12 days of
sliding, and successfully colonized. Liu H. et al. (2021) found a similar
situation when studying the colonization of poplar rhizosphere by149

Pseudomonas fluorescens. This study did not verify the colonization of
the inoculated strains, but from the sequencing results, the PSB did not
become the dominant population (except A2, where Enterobacteriaceae
is the dominant population).
Although PSB did not become the dominant population and did notaffect bacterial diversity, it still affected the structure of the rhizospherebacterial community, which was consistent with what Huang et al. (2020)
found when studying the growth promoting effect of Bacillus on peanuts.
In this study, the bacterial community structure of inoculated anduninoculated soil was different, and Flavisolibactor, which became thedominant species in F and G treatments, had a close relationship with soil
ACPase activity. Liu et al. (2020) also found that although exogenous
PSB did not become the dominant species in tomato rhizosphere, itchanged the soil bacterial community structure, caused the decline ofrelative abundance of some indigenous bacteria, and also increased theabundance of several bacteria. That study also found that soil properties
had a greater impact on soil microbial community. Liu believed that PSB
changed soil properties and rhizosphere microbial communities bysecreting organic acids. This is similar to our research results.

6.4 Conclusion and Perspectives
The diversity of global soils makes it appropriate to manage P
150

fertilization differently (Mengel, 1997). Results of our study supported
this view. As findings obtained from our study suggest, a same strain’s
efficiencies in releasing soil P and improving labile P differ in different
soil types (La and Ci). These differences might be partly attributable tothe influence of the original soil P fractionation. P is assigned to different
fractions during the conversion process. As for La, which is rich in ironand aluminum, is more likely to bind P to the NaOH-P fraction, leading toa low labile P situation. My study showed that the combination of TCP
and PSB in La produced much higher labile P and maize seedling P
accumulation than one treatment alone, whereas the combination of themin Ci did not produce a significant increase in the maize seedling P
accumulation compared to PSB alone. In general, in La, additional P isbeneficial for increasing the maize seedling P accumulation. Its use incombination with PSB can further optimize planting effects, whereas inCi, using PSB to mobilize the soil P can produce good results.
As Barrow (2022) pointed out, long-term fertilized soils havealready accumulated large amounts of P. What must be done is totransform it to useful nutrients for plants rather than to continue to add
extra P. PSB is promising to mobilize accumulated P in soils. Study of P
release by PSB in different soils will help to find PSB suitable fordifferent soil type, thus promoting the development of targeted andefficient PSB fertilizer and providing possible help for the sustainable151

development of agriculture.
Overall, this research examined the release of P by PSBs in two
typical soil types in China. The results demonstrated that the effect of
PSB inoculation on P release varied, with a primary increase in labile P
for Soil Type La and labile P for Soil Type Ci.
Furthermore, this research clarified that PSBs had a direct or indirect
influence on enhancing maize P accumulation by impacting soil
physicochemical indicators. The efficiency of this enhancement variedamong different soil types.
Moreover, this research investigated the impact of exogenous PSBs
on maize P accumulation in natural soil. The findings revealed that PSBs
not only influenced the soil rhizobacterial community but also established
an indirect positive relationship with maize P accumulation.
These results contribute to a better understanding of the role of PSBs
in P release and their effects on maize P accumulation in different soil
environments. Further research is warranted to explore the mechanisms
underlying these interactions and to optimize the application of PSBs for
improved nutrient management in agricultural systems.
In conclusion, this research demonstrated that PSB is an effectivemeans to mobilize P in soil. With the diverse soil profiles found globally,
including significant P retention in long-term cultivated fields, further
investigation into the dynamics of P release by PSB in various soil types
152can contribute to the more efficient utilization of PSB. I hope that myresearch provides valuable insights for the enhanced utilization of PSB.

153

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Acknowledgment
My heart is overflowing with gratitude as I reflect on the completionof this research. Without the careful guidance and unwavering support ofProfessor Wasaki Jun, Professor Nakatsubo, and Professor Ueda, I would
never have been able to achieve this milestone. Their mentorship has not
only enriched my work, but also my personal life. My heartfelt thanks goout to each of them.
I am also deeply grateful to Ms. Christiana, whose invaluableassistance was instrumental in the completion of my doctoral dissertation.
Her kindness and generosity will always be remembered.
As I think back on the long hours spent in the lab, I am reminded of
the guidance and support of Ms. Tateishi and Mr. Yamamura. Theirexpertise and dedication were crucial to the success of my experiments,
and I am forever indebted to them.
I would like to express my thanks to Xiong Zilin, Aihara, Abiko,
Yamada, Wang Ruixin, Saisako, and Lydia for their unwavering support.
Your contributions have been immeasurable, and I could not have done it
without you. Thank you from the bottom of my heart!
啥都不说了，我妈才是最伟大的！向我最亲爱的妈妈敬礼！妈您
辛苦了！
毛小豆！妈妈爱你！你是最棒的！今后也要继续一起加油哦！
感谢我亲爱的姐姐！
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Appendix
I. Abbreviation
Abbr.

Full form

ACPaseAcid phosphatase

AKPaseAlkaline phosphatase

Al-P

Aluminum phosphate

Ca-P

Calcium phosphate

Ci

Cinnamon soils

Fe-P

Iron phosphate

HCl-P

HCl extracted phosphorus

La

Lateritic red earths

NaHCO3-P

NaHCO3 extracted phosphorus

NaHCO3-Pi

NaHCO3 extracted inorganic phosphorus

NaHCO3-Po

NaHCO3 extracted organic phosphorus

NaOH-PNaOH extracted phosphorus

NaOH-PiNaOH extracted inorganic phosphorus

NaOH-PoNaOH extracted organic phosphorus

NPase

Neutral phosphatase

M labile P

Moderately labile phosphorus

Olsen-P

Available phosphorus in soil extracted via NaHCO3
solution using the method proposed by Olsen in 1954

O-P

Occluded phosphate

P

Phosphorus

PRP

Phosphorus retention potential

PSB

Phosphate solubilizing bacteria

Resin-P

Resin exchangeable phosphorus
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Abbr.
TCP

Full form
Tricalcium phosphate, Ca3(PO4)2

II. Culture media and solution·NBRIP g/L
Glucose,10; MgCl2·6H2O, 5; MgSO4·H2O, 0.25; KCl, 0.2; (NH4)2SO4,
0.1; Ca3(PO4)2, 5; Mix components with distilled or deionized water.
Autoclave at 115°C for 15 minutes.
·LB g/L
Tryptone,10; Yeast Extract, 5; NaCl, 10; Dissolve components in distilled
or deionized water.
For LB agar should be called LA add agar to a final concentration of
1.5%.
Autoclave at 121°C for 15 minutes.
·50% glycerol
Make the 50% glycerol solution by diluting 100% glycerol in pure water.
Autoclave at 121 ℃ for 15min.

III. Equipment·PH meterPHS-3C Leici Shanghai China with electrode E-201.

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IV. Measuring methods·Molybdenum blue method (Murphy and Riley, 1962)
Reagent:(1) Sulfuric acid (5N): Dilute 70 mL of concentrated sulfuric acid to
500 mL.
(2) Ammonium molybdate: Dissolve 20 g of high-purity ammonium
molybdate in water and dilute to 500 mL. Store the solution in a brownglass bottle.
(3) Ascorbic acid (0.1 M): Dissolve 1.32 g of ascorbic acid in 75 mL
of water. Ideally, this solution should be prepared on the day of use as
ascorbic acid is easily oxidized. If the solution needs to be stored, add 25
mg of disodium ethylenediaminetetraacetate and 0.5 mL of formic acidper 75 mL of solution to stabilize the ascorbic acid.
(4) Potassium antimony tartrate (274.3 mg of rhodium per mL):
Dissolve 0.2743 g of potassium antimony tartrate in distilled water anddilute to 100 mL.
(5) Reagent mixture: Mix 125 mL of 5 N sulfuric acid with 37.5 mLof ammonium molybdate, and then add 75 mL of ascorbic acid solution
and 12.5 mL of potassium antimony tartrate solution. This reagent should
be prepared as needed as it is stable for no more than 24 hours.
Stock phosphate solution: Prepare a solution containing 0.1757 g ofpotassium dihydrogen phosphate per liter. This solution contains 40 mg P
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(as phosphate) per liter. Phosphate working solution: Dilute the stock
solution to a solution containing 0.2 μg P (as phosphate) per mL.
Gradually dilute the phosphate use solution to prepare standard
curve samples. Take 40 mL of the standard curve samples and the test
samples, add 8 mL of the mixed reagent, make up to 50 mL in a
volumetric flask, and mix well. After 15 minutes, measure the absorbanceof the solution at 721 nm in a 1 cm cuvette.

170