A rhizosphere bacterium PLr56 of the phosphorus-enriched plant Polygonum lapathifolium and its application

Abstract

The present invention discloses a rhizosphere bacterium PLr56 of Rumex acetosella, a phosphorus-enriched plant, and its application, belonging to the technical field of microbiology. This strain is Enterobacter chuanxiongensis Enterobacter chuandaensis PLr56, which was deposited at the General Microbiology Center of the China Committee for Culture Collection of Microorganisms on July 25, 2024, with the deposit number CGMCC NO: 31461. It is a rhizosphere bacterium with the functions of adapting to low available phosphorus, tolerating high available phosphorus, and promoting plant growth. Due to its growth-promoting abilities such as dissolving tricalcium phosphate and calcium phytate, nitrogen fixation, secreting indole acetic acid, and producing siderophores, it can promote plant growth and the accumulation of nitrogen and phosphorus in plants.

Description

Technical Field

[0001] The present invention belongs to the technical field of microbiology. Specifically, it is a rhizosphere bacterium of the phosphorus-enriched plant Polygonum lapathifolium L. Enterobacter chuandaensis PLr56 and its applications. Background Art

[0002] In order to ensure high and stable yields in the agricultural and forestry systems, a large amount of phosphate fertilizers have been continuously input during production activities. However, the seasonal utilization efficiency of phosphate fertilizers is low, and phosphorus in the soil is easily fixed, resulting in a rich soil phosphorus pool but low biological availability of phosphorus. The excess phosphorus that has not been utilized by organisms easily flows into water bodies, causing environmental problems such as water eutrophication. Utilizing phosphorus-enriched plants to absorb and enrich excess phosphorus in the soil can intercept phosphorus loss from the source, thereby reducing the negative impact of phosphorus on the environment.

[0003] Polygonum lapathifolium L. is an amphibious plant with a fast growth rate and strong environmental adaptability. Under high-phosphorus treatment, Polygonum lapathifolium L. has obvious advantages in phosphorus enrichment ability. Rhizosphere-promoting bacteria and plants have formed a highly close mutual relationship in the nutrient cycling processes such as nitrogen and phosphorus. They may play an important role in the phosphorus enrichment process of Polygonum lapathifolium L. Currently, the research on phosphorus-solubilizing and growth-promoting bacteria is very active. However, previous research on phosphorus-solubilizing and growth-promoting bacteria has mainly focused on their application in low-phosphorus soils. However, there is very little research on growth-promoting bacteria that can not only activate insoluble phosphorus in the soil under low available phosphorus conditions, improve the availability of phosphorus, but also promote the phosphorus cycle in the soil and enhance the phosphorus uptake by roots under high available phosphorus conditions.

[0004] Enterobacter chuanxiongensis ( Enterobacter chuandaensis ) is a newly discovered bacterial strain. Currently, it is found to have value in the diagnosis and treatment of infections, but its application in the field of agricultural resources and environment has not been reported yet. Bacteria have a short culture cycle and a fast growth rate, and can greenly and efficiently improve the extraction ability of phosphorus-enriched plants for excess phosphorus in the soil. Then, the phosphorus-enriched plants with high extraction ability are prepared into biochar and applied to the soil instead of phosphate fertilizers, forming an internal phosphorus recycling system in the agricultural ecosystem. This can not only reduce the input of phosphate fertilizers, alleviate the global phosphorus depletion crisis, but also reduce the environmental risks brought by excess phosphorus. Summary of the Invention

[0005] In order to explore and screen rhizosphere-promoting bacterial strain resources that can adapt to low available phosphorus environments, tolerate high available phosphorus environments, and improve the phosphorus accumulation ability of phosphorus-enriched plants, and apply them to low available phosphorus and phosphorus-excess soils, thereby improving the utilization rate of phosphate fertilizers and reducing the environmental risks caused by phosphorus-excess soils, the present invention provides a rhizosphere bacterium PLr56 of the phosphorus-enriched plant Polygonum lapathifolium L. This strain is a rhizosphere bacterium with the functions of adapting to low phosphorus, tolerating high phosphorus, and promoting plant growth. At the same time, the present invention also provides the application of this strain in plant growth promotion and the application in the preparation of a phosphorus-solubilizing and growth-promoting bacterial agent for Polygonum lapathifolium L.

[0006] The present invention is achieved through the following technical solutions: A rhizosphere bacterium PLr56 of the phosphorus-enriched plant Polygonum lapathifolium L., and this strain is: Enterobacter chuanxiongensis Enterobacter chuandaensis PLr56 was deposited at the General Microbiological Center of the China Committee for Culture Collection of Microorganisms on July 25, 2024. Its deposit number is CGMCC NO: 31461, and the address of the deposit unit is No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing

[0007] The said strain was isolated from the rhizosphere soil of Polygonum lapathifolium L

[0008] Through 16S rRNA gene and phylogenetic analysis, it can be known that the strain PLr56 has a 99.10% homology with the 16S rRNA nucleotide sequence of Enterobacter chuanxiongensis Enterobacter chuandaensis strain 090028. Therefore, the strain PLr56 was identified as Enterobacter chuanxiongensis Enterobacter chuandaensis 。

[0009] As the application of the above-mentioned rhizosphere bacterium PLr56 of the phosphorus-enriched plant Polygonum lapathifolium L. in plant growth promotion, the said plant growth promotion includes increasing the accumulation of phosphorus and nitrogen in plants and promoting the growth and development of plants

[0010] The said plants include Polygonum lapathifolium L

[0011] The said increase in the accumulation of phosphorus and nitrogen in plants includes increasing the accumulation of phosphorus and nitrogen in the above-ground and underground parts of plants

[0012] As the application of the above-mentioned rhizosphere bacterium PLr56 of the phosphorus-enriched plant Polygonum lapathifolium L. in the preparation of a phosphorus-solubilizing and growth-promoting bactericide for Polygonum lapathifolium L

[0013] Compared with the prior art, the present invention has the following advantages and beneficial effects

[0014] (1) The strain PLr56 involved in the present invention was first screened and isolated from the rhizosphere soil of Polygonum lapathifolium L. Because it has the growth-promoting abilities such as dissolving tricalcium phosphate and calcium phytate, nitrogen fixation, secreting indole acetic acid and producing siderophores, it can promote plant growth and the accumulation of nitrogen and phosphorus in plants

[0015] (2) By applying the PLr56 strain to the phosphorus-enriched plant Polygonum lapathifolium L., the present invention not only improves the ability of Polygonum lapathifolium L. to extract excess phosphorus in a high-available phosphorus environment, but also enhances the activation and utilization of insoluble phosphorus by Polygonum lapathifolium L. in a low-available phosphorus environment, and has good application potential in agricultural production

[0016] (3) The present invention can increase the aboveground biomass, phosphorus accumulation and nitrogen accumulation of Rumex sylvestris by inoculating Rumex sylvestris with the PLr56 strain. Soil culture experiments have shown that, compared with the control (Rumex sylvestris not inoculated with the PLr56 strain), the aboveground biomass, phosphorus accumulation and nitrogen accumulation of Rumex sylvestris inoculated with the PLr56 strain under potassium dihydrogen phosphate treatment are 2.30, 2.24 and 2.03 times of those of the control, respectively; the aboveground biomass and nitrogen accumulation of Rumex sylvestris inoculated with tricalcium phosphate are 1.59 and 1.62 times of those of the control, respectively; and the aboveground biomass, phosphorus accumulation and nitrogen accumulation of Rumex sylvestris inoculated with calcium phytate are 3.43, 3.54 and 2.82 times of those of the control, respectively. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] Figure 1 Phylogenetic tree of PLr56 strain constructed based on 16S rRNA gene sequence.

[0018] Figure 2 It is the phosphate dissolution zone control of PLr56 strain on NBRIP solid medium.

[0019] Figure 3 The growth status of PLr56 strain on Ashby nitrogen-free solid medium.

[0020] Figure 4 This is a graph showing the effect of the PLr56 strain in promoting the growth and nitrogen and phosphorus accumulation of Polygonum aviculare in soil culture experiments. DETAILED DESCRIPTION

[0021] The present invention is further described in detail below in conjunction with examples, but the embodiments of the present invention are not limited thereto.

[0022] Example 1: Isolation and identification of strains

[0023] (1) Collection of Polygonum aviculare

[0024] Collect Rumex sylvestris grown under high phosphorus treatment. Keep the integrity of the above-ground part of the plant during collection. First, dig out the surface soil, then take out the roots of the Rumex sylvestris along its growth direction, put the whole plant into a sterile ziplock bag and store it in a sampling box with ice packs, and then bring it back to the laboratory for isolation of bacterial strains.

[0025] (2) Strain screening and isolation

[0026] The rhizosphere soil attached to the roots was collected by shaking the soil on a clean bench. 1 g of rhizosphere soil was weighed and added to a test tube containing 9 mL of 0.85% sterile saline. After sufficient shaking, 10 dilutions were prepared in sequence according to the dilution gradient method. -4 , 10 -5 and 10 -6The rhizosphere soil diluent of the gradient was taken, and 100 µL was inoculated on LB and NA media respectively. Three replicates were made for each gradient and each medium. The culture was carried out at a constant temperature of 28 °C. After 3 days, the colonies that grew vigorously and had a complete morphology on the medium were counted and screened for purification. The medium used for purification was the same as the separation medium. The purified single colonies were inoculated into LB liquid medium, and an appropriate amount of the bacterial solution was stored in glycerol at -80 °C.

[0027] (3)Strain identification

[0028] The 16S rRNA gene sequence of the bacteria was analyzed using the bacterial universal primers 27F (5'-AGAGTTTGATCCTGGCTCAG-3') and 1492R (5'-GGTTACCTTGTTACGACTT-3'). The sequence was determined by Shanghai Sangon Biotech Co., Ltd. The sequencing results of the strain were subjected to homology comparison by Blast in NCBI. The MEGA7.0 software was used to construct a phylogenetic tree by the N-J method to determine the taxonomic status of the bacteria. See Figure 1 as shown.

[0029] The bacterium PLr56 was isolated from the rhizosphere soil of Polygonum lapathifolium L. Its 16S rRNA gene sequence was analyzed, and a BLAST homology comparison was performed in the NCBI database. This strain had a similarity of 99.10% with Enterobacter chuanxiongensis Enterobacter chuandaensis . PLr56 was identified as Enterobacter chuanxiongensis Enterobacter chuandaensis .

[0030] Therefore, in this example, after isolating, culturing, screening, purifying, and identifying the rhizosphere bacteria of Polygonum lapathifolium L. growing under high phosphorus treatment, the rhizosphere bacterium PLr56 of the phosphorus-enriched plant Polygonum lapathifolium L. can be obtained Enterobacter chuandaensis PLr56.

[0031] Example 2: Detection of the growth-promoting function of the strain

[0032] The strain PLr56 obtained in Example 1 was used for the following detection of growth-promoting functions:

[0033] (1)Detection of phosphorus solubilization ability

[0034] The test bacteria were inoculated on the NBRIP solid medium with tricalcium phosphate or calcium phytate as the sole phosphorus source by the spot inoculation method. The strain was cultured in an incubator at 28 °C for 7 days. The strain with a phosphorus solubilization circle was the phosphorus-solubilizing bacterium. See Figure 2 as shown. Figure 2 The left figure in Figure 2 is the NBRIP solid medium with tricalcium phosphate as the sole phosphorus source, and the right figure in

[0035] From Figure 2 It can be seen that the colonies of strain PLr56 have a phosphorus-dissolving circle on both solid media, indicating its ability to dissolve inorganic insoluble phosphorus tricalcium phosphate and organic insoluble phosphorus calcium phytate.

[0036] After measuring the colony diameter and the phosphorus-dissolving circle diameter, calculate the phosphorus-dissolving index, that is: Phosphorus-dissolving index = Phosphorus-dissolving circle diameter / Colony diameter.

[0037] Take the bacterial suspension and inoculate it into centrifuge tubes containing NBRIP culture medium with tricalcium phosphate or calcium phytate respectively, with 3 replicates. The corresponding phosphorus source culture medium without inoculation is used as the control. Place it at 28 °C and 180 r·min -1 After shaking culture for 7 days, centrifuge the bacterial solution, and the culture supernatant is used for the determination of phosphorus concentration. The molybdenum blue colorimetric method is used to determine the available phosphorus concentration in the culture solution. Take 100 μL of the supernatant and add 2.5 mL of molybdenum antimony anti-color reagent, make up the volume to 25 mL and mix well. Color at room temperature for 30 min, and measure the absorbance value at 882 nm. Make a standard curve in the same way with the phosphorus standard solution, and calculate the available phosphorus concentration in the culture solution, that is, the phosphorus-dissolving amount.

[0038] After the above tests, the phosphorus-dissolving index and phosphorus-dissolving amount of strain PLr56 are shown in Table 1.

[0039] (2) Detection of nitrogen fixation ability

[0040] Use the cross-streaking method to inoculate the test bacterial strain on Ashby nitrogen-free solid medium, and incubate it at a constant temperature of 28 °C in an incubator for 7 days. The test strain that can grow on the medium is the nitrogen-fixing strain, see Figure 3 as shown.

[0041] From Figure 3 It can be seen that strain PLr56 grows well, indicating that this strain has a certain nitrogen fixation ability.

[0042] Take the bacterial suspension and inoculate it into centrifuge tubes containing Ashby nitrogen-free culture solution, with 3 replicates. The culture solution without inoculation is used as the control. Place it at 28 °C and 180 r·min -1 After shaking culture for 2 days, centrifuge, and take the supernatant to measure the nitrogenase activity. Use the bacterial nitrogenase ELISA detection kit to detect the nitrogenase. Add the culture solution and the HRP-labeled detection antibody to the coated microplate pre-coated with the nitrogenase antibody in turn, incubate and wash thoroughly. Color with the substrate TMB, and measure the absorbance value at a wavelength of 450 nm. Make a standard curve in the same way with the standard product, and calculate the nitrogenase activity in the culture solution.

[0043] After the above tests, the nitrogenase activity of strain PLr56 is shown in Table 1.

[0044] (3) Detection of indole acetic acid secretion ability

[0045] Take the bacterial suspension and inoculate it into a centrifuge tube containing SMS culture medium with 0.5 mg·mL -1 tryptophan. Repeat three times, and the culture medium without inoculation serves as a control. Place it in an incubator at 28 °C with shaking at 180 r·min -1 for 2 days, then centrifuge. Take the supernatant and measure the indole-3-acetic acid (IAA) concentration. The IAA concentration in the culture medium is determined by the Salkowski colorimetric method. Take 1 mL of the supernatant and add 2 mL of the chromogenic reagent (2 mL of 0.5 mol·L -1 FeCl3 + 98 mL of 35% (v / v) HClO4), mix well. After a dark reaction for 25 min at room temperature, measure the absorbance at 530 nm. Use the IAA standard solution to make a standard curve in the same way, and calculate the IAA concentration in the culture medium.

[0046] After the above tests, the IAA secretion of strain PLr56 is shown in Table 1.

[0047] (4)Siderophore production detection

[0048] Take the bacterial suspension and inoculate it into a centrifuge tube containing Chrome Azurol S (CAS) culture medium. Repeat three times, and the culture medium without inoculation serves as a control. Place it in an incubator at 28 °C with shaking at 180 r·min -1 for 2 days, then centrifuge. Take the supernatant and measure the relative content of siderophores. The relative content of siderophores in the culture medium is determined by the CAS method. Take 3 mL of the supernatant and add an equal volume of the CAS detection solution, mix well. Let it stand in the dark for 30 min, and measure the absorbance value As at 630 nm. Take another blank medium and mix it with an equal volume of the CAS detection solution, and use its absorbance value as the reference ratio Ar to calculate the relative content of siderophores.

[0049] After the above tests, the siderophore production of strain PLr56 is shown in Table 1.

[0050] Table 1 Detection data of growth-promoting functions of strain PLr56

[0051]

[0052] As can be seen from Table 1, the growth-promoting rhizobacterium Enterobacter chuandaensis PLr56 of Polygonum lapathifolium has a dissolution index and dissolution amount of tricalcium phosphate of 1.42 and 136.41 mg·L -1 , and a dissolution index of calcium phytate of 2.54 and 375.61 mg·L -1 , showing strong phosphorus-solubilizing ability. At the same time, it has the abilities of nitrogen fixation, indole-3-acetic acid secretion, and siderophore production. Therefore, the growth-promoting rhizobacterium Enterobacter chuandaensis PLr56 of Polygonum lapathifolium has good growth-promoting functions and has the potential for further development and application.

[0053] Example 3: Application of the Strain

[0054] Two groups of control experiments with inoculation and non-inoculation treatments were set up. The phosphorus concentration was 300 mg·P·kg -1 , and the phosphorus sources were potassium dihydrogen phosphate (soluble phosphorus), tricalcium phosphate (insoluble inorganic phosphorus), and calcium phytate (insoluble organic phosphorus). 100 mg·N·kg of urea was applied to each pot -1 , and each treatment was repeated 5 times.

[0055] A soil culture experiment was carried out. The air-dried and crushed soil was sieved and filled into pots. The pot size was 10 cm in inner diameter and 9 cm in height. Each pot was filled with 500 g of soil. After applying the corresponding phosphorus source, it was thoroughly mixed. Except for phosphorus, an appropriate amount of potassium sulfate was applied to each treatment to balance the potassium brought into the soil due to the application of potassium dihydrogen phosphate. After aging for 4 weeks, it was sterilized by γ-ray irradiation, and the minimum absorbed dose was 25.0 kGy. After sterilization, the available phosphorus contents in the soils treated with the phosphorus sources potassium dihydrogen phosphate, tricalcium phosphate, and calcium phytate were 183.37, 11.85, and 97.21 mg·kg -1 .

[0056] The seeds were directly sown in the sterilized soil. At the three-leaf stage, seedlings with good growth conditions and consistent growth trends were selected for thinning, with 1 plant per pot. When the plant height was about 5 cm, 15 mL of the prepared bacterial suspension and 25 mL of sterilized water were mixed and poured around the roots of the soil, while an equal amount of sterilized water was added in the non-inoculation treatment.

[0057] Natural light was used for illumination, and water was added irregularly according to the soil moisture condition of the potted plants. Samples were collected 3 weeks after inoculation. The plants were first rinsed with tap water and then rinsed with distilled water. After being washed and dried, they were divided into aboveground and underground parts. Some samples were bagged, blanched, and dried to a constant weight, and the dry weight was weighed and crushed for the determination of nitrogen and phosphorus contents. The plant biomass was determined by the oven-drying and weighing method. The plant phosphorus content was determined by the H2SO4-H2O2 digestion-molybdenum antimony anti-colorimetric method. The plant nitrogen content was determined by the H2SO4-H2O2 digestion-indigo colorimetric method.

[0058] The results of the soil culture experiment are shown in Figure 4 and Tables 2, 3, and 4 (Note: Different lowercase letters in the same column in Tables 2 to 4 indicate significant differences between different treatments at the 0.05 level).

[0059] As shown by Figure 4 , in the three rows of phenotypic diagrams from top to bottom, the phosphorus sources are potassium dihydrogen phosphate, tricalcium phosphate, and calcium phytate respectively. The left column is the non-inoculation condition, and the right column is the inoculation of PLr56. After inoculating PLr56 under each phosphorus source treatment, the plant height, number of branches, and number of leaves of Polygonum lapathifolium L. increased significantly.

[0060] Table 2 Effects of Inoculating PLr56 Strain on the Biomass of Polygonum lapathifolium L.

[0061]

[0062] Table 3 Effects of inoculating strain PLr56 on phosphorus accumulation in Polygonum lapathifolium

[0063]

[0064] Table 4 Effects of inoculating strain PLr56 on nitrogen accumulation in Polygonum lapathifolium

[0065]

[0066] The strain PLr56 was re-inoculated into the rhizosphere soil of Polygonum lapathifolium to explore its regulatory effect on the phosphorus accumulation ability of Polygonum lapathifolium. Biomass is one of the important indicators for evaluating the growth-promoting ability of strains and is also one of the important indicators for plant environmental adaptability. Phytoremediation technology usually aims to transfer and remove toxic or excessive elements in the soil by mowing and harvesting the above-ground parts. Therefore, the above-ground part is the key part for evaluating the absorption and enrichment ability of Polygonum lapathifolium for excessive phosphorus in the soil, and the phosphorus accumulation in the above-ground part is an important indicator for evaluating the ability of Polygonum lapathifolium to extract excessive phosphorus from the soil. In this experiment, rhizosphere growth-promoting bacteria were inoculated by root irrigation. The root system is the first part of Polygonum lapathifolium to contact the rhizosphere growth-promoting bacteria, and at the same time it is an important part for nitrogen and phosphorus absorption. Therefore, it is very necessary to analyze the response characteristics of the root system to rhizosphere growth-promoting bacteria.

[0067] As shown in Table 2, in terms of biomass, under the potassium dihydrogen phosphate treatment, the biomass of the underground and above-ground parts of Polygonum lapathifolium increased after inoculating strain PLr56, and the above-ground biomass was 2.30 times that of the non-inoculated; under the tricalcium phosphate treatment, the underground biomass of Polygonum lapathifolium did not change significantly after inoculating strain PLr56, while the above-ground biomass increased after PLr56 inoculation and was 1.59 times that of the non-inoculated; under the calcium phytate treatment, the biomass of the underground and above-ground parts of Polygonum lapathifolium increased after PLr56 inoculation, and the above-ground biomass was 3.43 times that of the non-inoculated.

[0068] As shown in Table 3, in terms of phosphorus accumulation, except for the tricalcium phosphate treatment, compared with the non-inoculated, the phosphorus accumulation in the above-ground and underground parts of Polygonum lapathifolium increased significantly after inoculating strain PLr56. Specifically, under the potassium dihydrogen phosphate and calcium phytate treatments, the phosphorus accumulation in the above-ground part of Polygonum lapathifolium was 2.24 times and 3.54 times that of the non-inoculated, respectively.

[0069] The effect of nitrogen on plant growth and development is very obvious. The tested strains have both phosphorus-solubilizing and nitrogen-fixing properties, and the absorption of nitrogen and phosphorus by plant roots often has a synergistic effect. Therefore, in addition to phosphorus accumulation, it is also necessary to analyze the effect of inoculating rhizosphere growth-promoting bacteria on the nitrogen accumulation of Polygonum lapathifolium.

[0070] As shown in Table 4, compared with non-inoculation, the nitrogen accumulation in the above-ground and below-ground parts of Polygonum lapathifolium L. significantly increased after inoculation with strain PLr56. Specifically, under the treatments of potassium dihydrogen phosphate, tricalcium phosphate, and calcium phytate, the nitrogen accumulation in the above-ground part of Polygonum lapathifolium L. was 2.03 times, 1.62 times, and 2.82 times that of non-inoculation, respectively. Therefore, inoculation with strain PLr56 can not only promote the growth of Polygonum lapathifolium L., but also improve its nitrogen and phosphorus accumulation ability.

[0071] The above are only the preferred embodiments of the present invention, and do not impose any form of limitation on the present invention. Any simple modification or equivalent change made to the above embodiments based on the technical essence of the present invention shall fall within the protection scope of the present invention.

Claims

1. A rhizosphere bacterium PLr56 of the phosphorus-enriched plant Polygonum lapathifolium L., characterized in that: The rhizosphere bacterium PLr56 of the phosphorus-enriched plant Polygonum lapathifolium is Enterobacter chuanxiongense Enterobacter chuandaensis PLr56, which was isolated from the rhizosphere soil of Polygonum lapathifolium, was deposited in the China General Microbiological Culture Collection Center on July 25, 2024, and its deposit number is CGMCC NO: 31461.

2. Use of the rhizosphere bacterium PLr56 of the phosphorus-enriched plant Polygonum lapathifolium L. according to claim 1 in promoting plant growth, characterized in that: The plant growth promotion includes: (1) Increasing the phosphorus accumulation and nitrogen accumulation of Polygonum lapathifolium; (2) Having the ability to dissolve tricalcium phosphate and calcium phytate; and, (3) Having the ability to secrete indoleacetic acid and produce siderophores.

3. The application according to claim 2, wherein: The increase in the phosphorus accumulation and nitrogen accumulation of Polygonum lapathifolium includes increasing the phosphorus accumulation and nitrogen accumulation in the above-ground and underground parts of Polygonum lapathifolium.

4. Use of the rhizosphere bacterium PLr56 of the phosphorus-enriched plant Polygonum lapathifolium L. as claimed in claim 1 in a phosphate-solubilizing and growth-promoting microbial inoculum, characterized in that: The phosphate-solubilizing and growth-promoting bacterium agent can improve the ability of Polygonum lapathifolium to extract excess phosphorus in a high-available phosphorus environment and the ability of Polygonum lapathifolium to activate and utilize insoluble phosphorus in a low-available phosphorus environment.

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