AU2021105222A4 - Application of Sophora flavescens Rhizospere Ensifer adhaerens SIP20-2 in Promoting the Growth of Maize - Google Patents

Abstract

The invention discloses the application of Sophora flavescens rhizospere Ensifer adhaerens SIP20-2 in promoting the growth of maize, and belongs to the technical field of microorganisms. The present invention isolates a Ensifer adhaerens SIP20-2 from the rhizosphere soil of Sophora flavescens. The bacterium has a strong ability to solubilize inorganic phosphorus, and also has the ability to produce IAA, siderophore, ACC deaminase, etc.. The Ensifer adhaerens SIP20-2 was made into an inoculum to inoculate maize seedlings. The results showed that the inoculum can significantly promote the growth of maize seedlings, especially the development of roots. Therefore, the present invention provides excellent strain resources for future development of microbial fertilizers for maize and other crops.

Description

Application of Sophoraflavescens Rhizospere Ensifer adhaerens SIP20-2 in

Promoting the Growth of Maize

TECHNICAL FIELD

The present invention relates to the technical field of microorganisms, in particular to the

application of Sophoraflavescensrhizosphere Ensifer adhaerensSIP20-2 in promoting the

growth of maize.

BACKGROUND

Phosphorus is one of the essential nutrient elements in the process of plant growth and

development, accounting for about 0.2% of the plant's dry weight. It is a component of

important substances such as nucleic acid, ATP and phospholipids. Therefore, phosphorus

participates in some important enzyme reactions and metabolic pathways etc. in plants. The

phosphorus used by plants is mainly derived from soil, and its existence is divided into

organic and inorganic forms. Although the total content of phosphorus in the soil is high

(400~1200mg/kg), the bioavailability of plants is less than 1%. Most phosphorus is in a

fixed state that is difficult for plants to directly absorb and utilize, and cannot meet the

growth and development needs of plants. Therefore, phosphorus is a nutrient that restricts

agricultural production on a global scale.

As an important part of the soil ecosystem, the type and quantity of microorganisms have

a great impact on the soil. Among them, the microorganisms that settle in the rhizome layer

and/or endogenous invasive plant tissues will have various beneficial effects on the plant

host. Those microorganisms with the characteristics above are called Plant Growth

Promoting Rhizobacteria (PGPR). PGPR was first proposed by Kloepper et al. (Kloepper,

1978). PGPRs that have been reported include Burkholderia and Agrobacterium,

Pseudomonas, Achromobacter, Bacillus, Enterobacter, Spirospira, Klebsiella And

Serratia and so on. However, there are few reports on the medicinal plant Sophora

flavescens PGPR (Lei Haiying, 2020).

The growth-promoting effect of PGPR can directly or indirectly affect the growth of plants

through several different mechanisms. Among them, Phosphorous Solubilizing

Microorganisms (PSM) are an important subgroup, and their secretion of low molecular

weight organic acids is considered to be involved in phosphorus solubilization. The main

mechanism is to convert insoluble phosphorus in the form of P043 - into soluble phosphorus

that can be absorbed and utilized by plants in the form of HP04 2- and H2P04-, providing

plants with more available phosphorus, thereby increasing the utilization of insoluble

phosphorus rate and dissolve phosphorus efficiency, and promoting plant growth,

improving the soil environment, and promote agricultural production. Therefore, it is of

great significance to study the utilization of phosphorus solubilizing microorganisms.

Sophoraflavescens is a perennial medicinal plant. In order not to compete for land with

farmland, Sophoraflavescens is often planted on poor soil. Therefore, Sophoraflavescens

has a well-developed roots, which can fully absorb and utilize trace amounts of soluble

phosphorus in the soil. Therefore, the rhizosphere phosphate solubilizing PGPR isolated

from the rhizosphere microorganisms of triennial Sophora flavescens of the present

invention lays a foundation for promoting crops growth and further research and

development of microbial fertilizer.

SUMMARY

The purpose of the present invention is to provide the application of Sophoraflavescens

rhizosphere Ensifer adhaerens SIP20-2 in promoting the growth of maize, so as to solve the above-mentioned problems in the prior art. The strain has obvious growth-promoting effects on maize.

In order to achieve the objectives above, the present invention provides the following

solutions:

The invention provides the application of Ensifer adhaerens SIP20-2 in promoting the

growth of maize.

Preferably, the phosphate Ensifer adhaerens SIP20-2 is isolated from the rhizosphere soil

of Sophoraflavescens.

The invention also provides the application of the inoculum containing the Ensifer

adhaerens SIP20-2 in promoting the growth of maize.

The invention also provides the application of the bacterial fertilizer containing the Ensifer

adhaerens SIP20-2 in promoting the growth of maize.

The present invention discloses the following technical effects:

The Sophoraflavescens rhizosphere Ensifer adhaerens SIP20-2 of the present invention

has a strong ability to solubilize inorganic phosphorus, and at the same time has the abilities

of producing IAA, producing siderophore, and producing ACC deaminase.

The Sophoraflavescens rhizosphere Ensifer adhaerens SIP20-2 of the present invention is

made into an inoculant to inoculate maize seedlings. The results show that the inoculum

can obviously promote the growth of the roots of maize seedlings, especially the

development of the roots. Therefore, the present invention provides excellent strain

resources for the future development of microbial fertilizers for maize and other crops.

BRIEF DESCRIPTION OF THE FIGURES

In order to explain the embodiments of the present invention or the technical solutions in

the prior art more clearly, the following will briefly introduce the drawings that need to be

used in the embodiments. Obviously, the drawings in the following description are only

some embodiments of the present invention. For those of ordinary skill in the field, without

creative work, other drawings can be obtained based on these drawings.

Figure 1 shows the colony morphology and electron microscopy of the strain SIP20-2;

Figure 2 shows the growth curve of strain SIP20-2 in 24 hours;

Figure 3 shows the growth curve of strain SIP20-2 under different pH conditions;

Figure 4 is a phylogenetic tree of SIP20-2 strain based on 16S rDNA sequence;

Figure 5 is the qualitative determination result of the phosphate solubilizing ability of strain

SIP20-2;

Figure 6 shows the influence of CK and strain SIP20-2 on morphological growth index 1

of maize seedlings;

Figure 7 shows the influence of CK and strain SIP20-2 on morphological growth index 2

of maize seedlings;

Figure 8 is a diagram of maize roots growth after treatment with CK and strain SIP20-2.

DESCRIPTION OF THE INVENTION

Various exemplary embodiments of the present invention will now be described in detail.

The detailed description should not be considered as a limitation to the present invention,

but should be understood as a more detailed description of certain aspects, characteristics,

and embodiments of the present invention.

Embodiment 1 Isolation and identification of Ensifer adhaerens SIP20-2

1. Soil samples

Triennial Sophoraflavescens rhizosphere soil collected from Zhendong Chinese Herbal

Development Co., Ltd., Changzhi City, Shanxi Province.

2. Strain isolation and identification

For the soil suspension collected above, 200 L each was uniformly spread on the Monkina

inorganic phosphorus solid medium by the conventional gradient dilution method for

separation and screening, and a single colony strain SIP 20-1 was obtained (see Figure 1).

Observing the morphology of the isolated strain SIP 20-2 on solid LB medium (peptone 10

g, yeast extract 5 g, NaCl 10 g, distilled water 1 L, pH 7.0). It was found that the edge of

the colony was neat, smooth, spherical, white, opaque, and convex in the middle, with a

colony diameter of 2.31 mm (see Table 1).

Table 1 Morphological analysis of strain SIP 20-2

Sri GrmSurface Colony Mrhl Morholo Protrusion property Transparency diameter nber egtive/Positive Edge (mm) SIP20-2 Negative Neat Protsion in smooth opaque 2.31 Spherical

Through the Gram staining method (inoculate the activated strain on LB solid medium,

culture at 28°C for 2-3 days, and stain), it can be observed from the microscope that SIP

-2 cells are spherical and belong to Gram Negative.

Refer to the ManualforIdentification of Common BacterialSystems for physiological and

biochemical analysis of SIP 20-2. Strain SIP 20-2 starch hydrolysis test: inoculating the

strain into a starch medium, culturing at 28°C for 4 days, adding iodine solution dropwise

to observe that the colony has no transparent circle around and is negative; gelatin

liquefaction test is negative; phenylalanine deaminase test is negative; malonate test is

negative; methyl red test is positive; citrate, urease, and Voges-Proskauer tests were all negative (see Table 2). According to the growth ability of the strain, the 24 h growth curve of the strain SIP 20-2 was measured (see Figure 2). The test strain was in the logarithmic growth phase for the first 10 hours, and the subsequent growth was slow with little change; the optimum pH value of the measured strain was 5.5 in the 5.5-10.0 pH gradient (see

Figure 3).

Table 2 Determination of physiological and biochemical analysis of SIP20-2 strain

Starch Gelatin Phenylalanine Malonate Methyl red Citrate Urease Voges- Catalase Strain hydrolysis liquefaction deaminase test test test test Proskauer test test test test test

SIP20-2 - - - - - - -+

The bacterial 16S rRNA universal primers (16s27-F: 5'-AGAGTTTGATCCTGGCTCAG

3', 16s1492-R: 5'-GGTTACCTTGTTACGACTT-3') were selected for PCR amplification

of the test strain SIP 20-2, and the measured sequence ( as shown in sequence 1) was

compared in NCBI and GenBank database, and the Neighbor-Joining method in MEGA7.0

was used to construct a phylogenetic tree. The results showed that the strain SIP 20-2

belongs to Ensifer and is highly homologous to the strain Ensiferand its sequence similarity

with the sequence of Ensifer adhaerensCasidaA(T) is 99.71% (see Figure 4).

The sequence shown in sequence 1 is as follows:

ggcggcttgacatgattacgccagcttgcatgcctgcaggtcgacgattacggctaccttgttacgacttcaccccagtc gctgaccctaccgtggttagctgcctccttgcggttagcgcactaccttcgggtagaaccaactcccatggtgtgacggg cggtgtgtacaaggcccgggaacgtattcaccgcagcatgctgatctgcgattactagcgattccaacttcatgcactcg agttgcagagtgcaatccgaactgagatggcttttggagattagctcgacctcgcggtctcgctgcccactgtcaccacc attgtagcacgtgtgtagcccagcccgtaagggccatgaggacttgacgtcatccccaccttcctctcggcttatcaccg gcagtccccttagagtgcccaactaaatgctggcaactaagggcgagggttgcgctcgttgcgggacttaacccaacatc tcacgacacgagctgacgacagccatgcagcacctgtctccgatccagccgaactgaaggaaaacgtctccgtaatccgc gatcgggatgtcaagggctggtaaggttctgcgcgttgcttcgaattaaaccacatgctccaccgcttgtgcgggcccct gtcaattcctttgagttttaatcttgcgaccgtactccccaggcggaatgtttaatgcgttagctgcgccaccgaacagt aaactgcccgacggctaacattcatcgtttacggcgtggactaccagggtatctaatcctgtttgctccccacgctttcg cacctcagcgtcagtaatggaccagtgagccgccttcgccactggtgttcctccgaatatctacgaatttcacctctaca ctcggaattccactcacctcttccatactctagacacccagtatcaaaggcagttccggggttgagccccgggatttcac ccctgacttaaatgtccgcctacgtgcgctttacgcccagtaattccgaacaacgctagccccttcgtattaccgcgctg ctggcacgagttagccgggcttcttctcggtaccgtcattatcttcaccggtgaaagagctttacatc Embodiment 2 Determination of the growth-promoting ability of Ensifer adhaerensSIP20

21.

1.Test plants

Maize was selected as the test plants, and the commercial seed Xianyu 335 was used as the

maize seed.

2. Test soil

The composition ratio of the substrate soil used in the experiment is peat: perlite:

vermiculite=3:1:1.

3. Test method

3.1 Determination of the growth-promoting ability of the tested strain 20-2

3.1.1 Determination of phosphorus solubilization ability

Picking the test strain SIP 20-2 and inoculating it on a plate containing Ca3(PO4)2 insoluble

phosphate Montana solid medium (glucose 10 g, (NH4)2SO4 0.5 g, NaCl 3 g, KCl 0.3 g,

FeSO4) •7H20 0.03 g, MnSO4•H20 0.03 g, MgSO4•7H20 0.3 g, CaCO3 5 g, yeast extract

0.4 g, agar 20 g, distilled water 1000 mL, pH 7.0-7.5; inorganic phosphorus medium:

calcium phosphate 2 g/100 mL; Organophosphorus medium: lecithin 0.2 g/1000 mL),

cultured at 37°C for 3-4 days, observing the formation of the phosphate ring, recording and

calculating the ratio of the diameter of the phosphate ring to the colony diameter (D/d) .

3.1.2 Determination of IAA production capacity

The Salkowki colorimetric method (Feng Weiwei et al., 2016) was used to quantitatively

determine the ability of the test strain SIP 20-2 to produce IAA. The test strain SIP 20-2

was inoculated into king liquid medium (peptone 20.0 g, K2HPO4 1.725 g, C). Glycerol

15.0 mL, MgSO4•7H20 1.5 g, tryptophan 0.1 g, pH 7.2), performing shake culture at 28°C,

180 rpm/min for 2 days, then centrifugation of the culture solution at 5000 rpm/min, 4°C

for 3 min, taking the supernatant and the colorimetric solution (50 mL of H20, 1 mL of 0.5

mol/L FeCl3, 30 mL of concentrated sulfuric acid) and mixing them in an equal amount at

a ratio of 1:1, and leaving them in the dark for 30 minutes; with the culture medium as a

blank control, determining the absorbance value of the culture medium at a wavelength of

530 nm, 3 replicates for each sample, and calculating the IAA concentration of the culture

medium through the standard curve.

3.1.3 Determination of iron carrier production capacity

The test strain SIP 20-2 was inoculated into MKB medium (containing acid hydrolyzed

casein 5 g/L, glycerol 15 mL/L, K2HPO4 2.5 g/L, MgSO4•7H20 2.5 g/L), 30°C, 180

rpm/min; The strain SIP 20-2 underwent shake culture for more than 2 days, and was

centrifugated at 10000 rpm/min at 4C for 7 min. The supernatant 100 pL each of the

supernatant and the CAS detection solution (solution A: 0.07 g CAS, 50 mL deionized

water; solution B: 0.091 g HDTMA, 50 mL deionized water; solution C: 0.08 g FeCl3, 50

mL deionized water; 10 mL of solution C is slowly added to 50 mL of solution A, mixed

and slowly added to 40 mL of solution B, stirring while adding)were taken, and 20 mL of

% sucrose solution, 50 mL of 10% acid hydrolyzed casein, 30 mL of1mM MgSO4, agar

solution (7.2 g agar powder, 300 mL deionized water) were added; 4 mL of sucrose

solution, 12 mL of acid hydrolyzed phenol, 8 mL of MgSO4 solution, 40 mL of CAS dye solution, 40 mL of phosphate buffer were was added into the agar solution in order and mixed; sterile distilled water as a blank, reaction at room temperature for 1 h, recording the absorption value of the sample (As) and control sample (Ar) at the wavelength of 630 nm and entering the following formula to calculate the iron carrier unit:

Iron carrier unit (%)=(As -Ar)/Arx100%

3.1.4 Determination of ACC deaminase activity

QUsing 1 mL of Tris-HCl buffer with a concentration of 0.1 mol/L and pH 7.6 to

resuspend the bacteria in a 2 mL EP tube, and centrifuge for 5 min at 4 °C, 12000 r/min

@Using 600 L of Tris-HCl buffer with a concentration of 0.1 mol/L and pH 8.5 to

resuspend the bacteria; @Adding 30 L of toluene and shaking for 30 s immediately to

break the cells and obtain a bacterial suspension, which contains cell fragments; @In a 2

mL EP tube, adding 200 L of the cell extract above, then adding 20 L of ACC with a

concentration of 0.5 mol/L, mixing well, and then putiung it in a 30 °C water bath for 15

min; @In the reaction quickly adding 1 mL of HC solution with a concentration of 0.56

mol/L to the solution, mixing well, and terminating the reaction; @Centrifugation for 5

min at room temperature and 12000 r/min; (Adding the above-mentioned 1 mL of

supernatant in a 5 mL centrifuge tube, then adding 800 L of 0.56 mol/L HCl solution, and

mixing well; @Adding another 2% 2,4-dinitrophenylhydrazine solution 300 [L, and then

water bath in a water bath for 30 min at temperature of 30 °C; @After the reaction is over, adding 2 mL of NaOH solution with a concentration of 2 mol/L to carry out the color

reaction for 5 min; @When the color is stable, the absorption value at OD540m was

detected, and the blank control is sterile water; the absorption value of each sample is substituted into the standard curve of a-butyric acid, and the content of a-butyric acid is calculated (Zhang Guozhuang, 2014 ).

3.2 Bacterial inoculation pot experiment

3.2.1 Preparation of inoculum

Picking SIP 20-2 single colony into 5 mL liquid LB medium (NaC1 10 g/L, tryptone 10

g/L, yeast 5 g/L, agar 20 g/L, pH 7.0), 28°C, 180 rpm/min, carrying out overnight shaking

culture; adding 1% of the 5 mL overnight bacteria above to 50 mL LB and continuing

shaking culture for 4 h, measure OD600, and diluting the OD value to 0.5 to make the

effective number of bacteria reach 108 cfu/mL.

3.2.2 Pot experiment

The ratio of the substrate (sterilized) is peat: perlite: vermiculite = 3:1:1, and the blank

substrate (with the corresponding sterile water) as the control, each substrate 60 g, adding

24 mL of the dilution above. After mixing the bacteria liquid, setting 4 repetitions, 2 Xianyu

335 maize seeds for each repetition (needed to be soaked at 30°C one day in advance),

placing in a greenhouse at 25°C for constant temperature cultivation, and the light time is

12 h/d. The light intensity is about 2000 Lux.

3.2.3 Determination of Physiological Indexes of Maize Seedlings

After 21 days of growth of the corn seedlings, measuring the chlorophyll of the fully

expanded second leaf of the corn seedlings of the inoculated group and the control group

respectively, recording and measuring the plant height, stem thickness, leaf area, fresh

weight above ground, dry weight above ground, and underground fresh weight,

underground dry weight, root length, root thickness, root morphology and so on.

4. Results and analysis

4.1 The growth-promoting ability of the tested strain

The phosphate-dissolving transparent circle of the tested strain SIP20-2 (see Figure 5) has

a transparent circle-to-colony diameter ratio of 2.25, an IAA production capacity of 18.236

mg/L, a siderophore production capacity of 65.1%, and ACC deaminase specific activity

of 0.0025 U /mg (see Table 3).

Table 3 Determination of growth-promoting ability of strain SIP 20-2

ACC Phosphorus IAA S erophore Amount of a- deaminase Strain D/d solubilizing concentration contest % butyric acid specific ability ( pg/mL) (pmoL) activity (U-mg-') SIP20- 2.25 ++ 18.236 65.1 0.1406 0.0025 2

4.2 The effect of tested strain SIP 20-2 on maize growth

As shown in Table 4 and Figure 6, among the three indicators of plant height, leaf area,

and root length, except for the leaf area lower than the control group, the other two growth

indicators are higher than the control group; and the maize after inoculation with SIP20-2,

the index of seedling root thickness, fresh weight on the ground, underground fresh weight,

and underground dry weight (Figure 7), compared with the control group, is increased by

53.52%, 81.32%, 43.27% and 247.47%, respectively, indicating that the Ensifer adhaerens

SIP20-2 brings a significant effect in promoting the growth of maize. It can be seen from

Figure 8 that the maize inoculated with Ensifer adhaerens SIP 20-2 has a developed roots, more fibrous roots, and strong fixation ability, which can significantly improve the underground nutrient absorption of corn and the transportation of nutrients to the ground.

Table 4 The influence of CK and strain SIP 20-2 on the growth indexes of maize

seedlings

indexes SIP 20-2 CK Plant height (cm) 31.833+17.83cd 31.333+1.761cd Leaf area (cm2 ) 17.32±9.994d 27.856+1.706a Root length (cm) 29.9±5.086a 21.4±6.158a Stem thickness ( 2.076±0.047b 2.206±0.338a mm) Root thickness ( 1.463+0.237ab 0.953±0.208b mm) Fresh weight on the 2.823±0.261ab 1.557+0.562cd ground (g) Dry weight on th 0.252±0.035a 0.456±0.604a ground (g) Underground fresh 1.957±1.022a 1.366+0.173ab weight (g) Underground dry 0.549±0.675a 0.158±0.078a weight (g) Note: Different letters after the data in the table indicate significant differences (p<0.05).

The above-mentioned embodiments only describe the preferred mode of the present

invention, and do not limit the scope of the present invention. Without departing from the

design spirit of the present invention, various modifications and improvements made by those skilled in the field to the technical solution of the present invention should fall within the protection scope determined by the claims of the present invention.

Claims (4)

THE CLAIMS DEFINING THE INVENTION ARE AS FOLLOWS

1. Observation of Ensifer adhaerens SIP20-2 in promoting maize growth.

2. The application according to claim 1, wherein the Ensifer adhaerensSIP20-2 is isolated

from the rhizosphere soil of Sophoraflavescens.

3. Including the observation of an inoculum of the Ensifer adhaerens SIP20-2 described in

claim 1 or 2 in promoting the growth of maize.

4. Including the observation of bacterial fertilizer of Ensifer adhaerensSIP20-2 described

in claim 1 or 2 in promoting the growth of maize.