Phosphorus-solubilizing fungus xzy3psf and application thereof

Abstract

The application provides a kind of phosphorus-solubilizing fungus XZY3PSF, and belongs to the technical field of microorganism, the phosphorus-solubilizing fungus XZY3PSF is significantly higher than the reported phosphorus-solubilizing bacteria in the efficiency of dissolving phosphorus by more than 7 times, after 10 generations, it still has good phosphorus-solubilizing activity.The highest value of effective phosphorus content dissolved by XZY3PSF bacteria under different concentrations of tricalcium phosphate is between 500-570mg / L, and the effective phosphorus content in the medium after mineralization of calcium phytate is as high as 368.6mg / L, therefore, XZY3PSF bacteria can degrade organic phosphorus and inorganic phosphorus with high activity.At the same time, XZY3PSF bacteria have antagonistic effect on banana fusarium wilt pathogen and other soil-borne diseases, and the inhibition rate is 66.2%-77.0%.The phosphorus-solubilizing fungus XZY3PSF has both high-efficiency phosphorus-solubilizing effect and antagonistic effect on soil-borne diseases, and provides excellent microbial strains for the research and development of biological bacterial fertilizer and / or soil conditioner.

Description

TECHNICAL FIELD

[0001] The present application belongs to the field of microbial technology, and particularly relates to a phosphorus-solubilizing fungus XZY3PSF and application thereof. BACKGROUND

[0002] Phosphorus is one of the three essential nutrients for crop growth and development, and plays an important role in cell division, energy transfer, signal transduction, nucleic acid synthesis and photosynthesis of plants. It is also the most important limiting factor in agricultural production. Phosphorus is added to the soil in the form of phosphate fertilizer. According to statistics, the seasonal utilization rate of phosphate fertilizer in the soil is generally only 10% to 25% in China, and the part not utilized by plants is converted into insoluble fixed form. In acid soil, the insoluble inorganic phosphorus in the soil is mostly fixed as phosphoric acid iron aluminum compounds, and in neutral and calcareous soil, it is mostly fixed as phosphoric acid tricalcium compounds. Therefore, how to develop and utilize the soil-fixed phosphorus, improve the effective utilization rate of phosphorus in the soil, and reduce the application of chemical phosphorus fertilizer is one of the problems that must be solved in sustainable agriculture in China. Adjusting the soil pH, applying organic fertilizer, soil flooding treatment, reasonable application of phosphorus fertilizer or using phosphorus-solubilizing microorganisms to activate the insoluble phosphorus in the soil are the ways to improve the effectiveness of soil phosphorus, among which, using phosphorus-solubilizing microorganisms to improve the effectiveness of soil phosphorus not only meets the current action demand of "zero growth of chemical fertilizer use", but also can reduce the agricultural environmental pollution caused by excessive application of phosphorus fertilizer.

[0003] The solubilization of microorganisms to insoluble phosphorus fertilizer such as phosphate rock and its application in agriculture is becoming a research hotspot widely concerned by researchers at home and abroad. Many soil microorganisms including bacteria, fungi and actinomycetes can participate in the dissolution, transformation and migration process of insoluble phosphorus in soil, and through the processes of acidification, chelation and exchange reaction, the insoluble phosphate fixed in the soil is changed into a soluble form to facilitate plant absorption, thereby increasing crop yield. At present, a large number of studies have been carried out on the phosphorus-solubilizing characteristics and mechanisms of phosphorus-solubilizing bacteria, but the related research on phosphorus-solubilizing fungi is still less. The reported fungi mainly include Aspergillus, Penicillium, Trichoderma, Talaromyces, Cladosporium, Paecilomyces and the like, and other groups are rarely reported. Compared with bacteria, phosphorus-solubilizing fungi have stronger ability to produce organic acids, and the phosphorus-solubilizing efficiency of some species is significantly higher than that of bacteria, and the stability of the phosphorus-solubilizing activity in the process of subculture is also significantly better than that of bacteria. Therefore, finding more fungi groups with phosphorus-solubilizing function can provide more strain resources for enhancing the availability of phosphorus in soil or fertilizer. SUMMARY

[0004] Therefore, the application aims to provide a phosphorus-solubilizing fungus XZY3PSF and application thereof, which has high phosphorus-solubilizing capacity and obvious prevention and treatment effect on soil-borne diseases.

[0005] In order to achieve the above-mentioned application purposes, the application provides the following technical solutions.

[0006] The application provides a phosphorus-solubilizing fungus XZY3PSF, which is named Talaromyces purpureogenus in taxonomy, and the strain number is XZY3PSF, and is preserved in the Guangdong Microbial Culture Collection Center with a preservation number of GDMCC No:62750.

[0007] The application also provides application of the above-mentioned phosphorus-solubilizing fungus XZY3PSF in soil phosphorus solubilization.

[0008] The application also provides application of the above-mentioned phosphorus-solubilizing fungus XZY3PSF in fertilizer phosphorus solubilization.

[0009] The application also provides application of the above-mentioned phosphorus-solubilizing fungus XZY3PSF in inhibition of plant pathogenic fungi.

[0010] Preferably, a spore suspension of the phosphorus-solubilizing fungus XZY3PSF is used for soil phosphorus solubilization and / or fertilizer phosphorus solubilization.

[0011] Preferably, the spore number of the spore suspension is 5×10 6 ~ 10×10 6 spores / mL.

[0012] Preferably, the preparation method of the spore suspension comprises the following steps.

[0013] The mycelium of the phosphorus-solubilizing fungus XZY3PSF is inoculated into a culture medium, and is cultured at a constant temperature of 26-30 DEG C until spores grow, the spores are washed with sterile water, and filtration is performed to obtain the spore suspension.

[0014] Preferably, the culture medium comprises a PDA culture medium, and the PDA culture medium comprises the following components in parts by weight: potato 190-210 parts, glucose 15-25 parts, agar 15-20 parts, and water 900-1100 parts.

[0015] Compared with the prior art, the application has the following beneficial effects.

[0016] The application provides a phosphorus solubilizing fungus XZY3PSF, which is identified as Talaromyces purpureogenus based on morphological characteristics in combination with ITS and beta-tubulin gene sequences. Compared with the reported phosphorus solubilizing bacteria, the phosphorus solubilizing fungus XZY3PSF has a solubilizing efficiency which is more than 7 times higher than that of the reported phosphorus solubilizing bacteria, and the strain still has good phosphorus solubilizing activity after being passed for 10 generations. The effective phosphorus content of the phosphorus solubilizing fungus XZY3PSF dissolved under different concentrations of calcium phosphate is between 500 and 570 mg / L, and the phosphorus solubilizing fungus XZY3PSF has a sustained mineralization effect on calcium phytate, and the effective phosphorus content in the culture medium after mineralization of calcium phytate is as high as 368.6 mg / L. Therefore, the phosphorus solubilizing fungus XZY3PSF can highly and actively degrade organic phosphorus and inorganic phosphorus. Meanwhile, the phosphorus solubilizing fungus XZY3PSF has an antagonistic effect on banana wilt pathogen and other soil-borne diseases, and the inhibition rate of the phosphorus solubilizing fungus XZY3PSF on plant pathogenic fungi is 66.2% to 77.0%. The phosphorus solubilizing fungus XZY3PSF has both high-efficiency phosphorus solubilizing effect and antagonistic effect on soil-borne diseases, and provides an excellent microbial strain for research and development of biological bacterial fertilizer and / or soil conditioner.

[0017] Biological preservation information:

[0018] The phosphorus solubilizing fungus (Talaromyces purpureogenus) XZY3PSF is preserved in the Guangdong Microbial Culture Collection Center, and the preservation number is GDMCC No:62750, the preservation date is August 30, 2022, and the preservation address is No. 59, Building 5, 100, Martyrs' Road, Guangzhou. BRIEF DESCRIPTION OF DRAWINGS

[0019] Figure 1 : A is the phosphorus solubilizing fungus XZY3PSF in the 5th day of the phosphorus solubilizing situation in the Mengjin solid culture medium plate, and B is the phosphatase activity analysis result of the phosphorus solubilizing fungus XZY3PSF in the 7th day of the phosphorus solubilizing fungus XZY3PSF in the Mengjin solid culture medium plate;

[0020] Figure 2 : The colony morphology and spore chain of the phosphorus solubilizing fungus XZY3PSF in the 3rd day of PDA plate culture, A is the front of the colony, B is the back of the plate, C is the spore structure, and D is the long spore chain;

[0021] Figure 3 : The ITS+beta-tubulin gene sequence DNAStar ClustalV method comparison of the phosphorus solubilizing fungus XZY3PSF establishes a phylogenetic tree;

[0022] Figure 4 : The phosphorus solubilizing effect of the phosphorus solubilizing fungus XZY3PSF on different concentrations of calcium phosphate;

[0023] Figure 5 Phosphorus release effect of phosphorus solubilizing fungus XZY3PSF on tricalcium phosphate under different spore concentrations;

[0024] Figure 6 Phosphorus release effect of phosphorus solubilizing fungus XZY3PSF on different concentrations of calcium phytate;

[0025] Figure 7 Antagonistic results of phosphorus solubilizing fungus XZYSPSF on 5 plant pathogenic fungi, wherein A is B2 strain; B is Nbm-1 strain; C is YSSGa-2 strain; D: YSSGa-10 strain; E: YSSGa-3 strain; wherein, the left side of A, B, C, D and E is the control of B2, Nbm-1, YSSGa-2, YSSGa-10, YSSGa-3 strain respectively, and the right side is the antagonistic effect of phosphorus solubilizing fungus XZYSPSF on the corresponding plant pathogenic fungi; F: the antagonistic effect of bacillus velezensis X5 strain on banana wilt pathogen B2 strain, the left side is B2 strain, and the right side is the antagonistic effect of bacillus velezensis X5 strain on banana wilt pathogen B2 strain. DETAILED DESCRIPTION

[0026] The application provides a phosphorus solubilizing fungus XZY3PSF, which is named as Talaromyces purpureogenus, and the strain number is XZY3PSF, and is preserved in the Guangdong Microbial Culture Collection Center, and the preservation number is GDMCC No:62750.

[0027] In the application, the phosphorus solubilizing fungus XZY3PSF is screened from a soil sample, the soil sample is collected from Beibeng Township, Motuo County, Nyingchi City, Tibet Autonomous Region, the soil type is black soil, and the sampling point is the soil around corn roots.

[0028] In the application, the screened phosphorus solubilizing fungus XZY3PSF is identified based on morphological characteristics in combination with ITS and beta-tubulin gene sequences, and is identified as Talaromyces purpureogenus XZY3PSF.

[0029] In the application, the primary hypha of the phosphorus solubilizing fungus XZY3PSF is white and transparent, green spores are generated after 3d, are gathered in the center, the surrounding hypha is white, and the back of the plate shows orange red; after being cultured for 15d, the hypha almost grows on the whole plate, the spores are gray green, the back of the plate is deep red, the spore structure is 3 branches and verticillate, the spores are oval, the size is 3.16-3.69 mu m, and the spores are long chains.

[0030] The application further provides application of the phosphorus solubilizing fungus XZY3PSF in soil phosphorus solubilization.

[0031] The application further provides application of the phosphorus solubilizing fungus XZY3PSF in fertilizer phosphorus solubilization.

[0032] The application further provides application of the phosphorus solubilizing fungus XZY3PSF in inhibiting plant pathogenic bacteria.

[0033] In the application, the phosphorus solubilizing fungus XZY3PSF is used to prepare soil improver and biological fertilizer, which can not only improve the utilization rate of soil available phosphorus and increase the yield of crops, but also effectively reduce the loss of phosphorus, and the phosphorus solubilizing fungus XZY3PSF also has a bacteriostatic effect on plant pathogenic bacteria, with an inhibition rate of 66.2% to 77.0%. The phosphorus solubilizing fungus XZY3PSF can effectively degrade organic phosphorus including calcium phytate and / or inorganic phosphorus including tricalcium phosphate. The plant pathogenic bacteria include one or more of banana wilt pathogenic bacteria, banana fruit rot pathogenic bacteria and cherry stem blight pathogenic bacteria.

[0034] As a preferred embodiment, a spore suspension of the phosphorus solubilizing fungus XZY3PSF is used for soil phosphorus solubilization and / or fertilizer phosphorus solubilization. The number of spores in the spore suspension is preferably 5×10 6 ~ 10×10 6 spores / mL, and further preferably 6×10 6 ~ 8×10 6 spores / mL. The preparation method of the spore suspension preferably comprises the following steps: inoculating mycelium of the phosphorus solubilizing fungus XZY3PSF into a culture medium, incubating at 26-30°C until spores grow, washing the spores with sterile water, and filtering to obtain the spore suspension. The culture medium preferably comprises PDA culture medium, and the PDA culture medium preferably comprises the following components by weight: potato 190-210 parts, glucose 15-25 parts, agar 15-20 parts, and water 900-1100 parts.

[0035] In the application, the phosphorus solubilizing fungus XZY3PSF can be used in combination with other soil improvers or fertilizers, or can be used alone. The phosphorus solubilizing fungus XZY3PSF can be prepared into granules, powders, suspensions or solutions for soil phosphorus solubilization and / or fertilizer phosphorus solubilization.

[0036] The technical solutions provided by the application will be described in detail below in combination with examples, but they should not be understood as limiting the scope of protection of the application.

[0037] The data processing of the application adopts Excel 2010 and SPSS19.0 to analyze and process data. Multiple comparisons adopt LSD method, correlation analysis adopts Pearson method, and drawing is made by using Excel 2010 software.

[0038] In the application, the phosphorus solubilizing ability of the strain is the difference between the effective phosphorus concentration of the inoculated culture solution and the effective phosphorus concentration of the non-inoculated culture solution, and the average value of three repetitions is expressed in mg / L.

[0039] Example 1

[0040] 1. Test materials and methods

[0041] 1.1 Test materials

[0042] 1.1.1 Test soil: The soil sample was collected from Beibeng Township, Motuo County, Nyingchi City, Tibet Autonomous Region, and the soil type was black soil. The sampling point was the soil around the corn roots.

[0043] 1.1.2 Test strains: Phosphorus solubilizing strains LGJPJ (Bacillus velezensis), Bacillus velezensis strain X5 and banana wilt pathogen B2 strain were isolated and preserved by the Microbial Resource Research and Utilization Research Group of the Institute of Environmental and Plant Protection, Chinese Academy of Tropical Agricultural Sciences.

[0044] 1.1.3 Test medium: (1) Simplified inorganic phosphorus solid medium: glucose 10.0 g, ammonium sulfate 2.0 g, tricalcium phosphate 5.0 g, agar 20.0 g, water 1000 mL, pH 7.00-7.50; (2) Simplified Mengjina solid medium: glucose 10.0 g, ammonium sulfate 2.0 g, calcium phytate 2.0 g, agar 20.0 g, water 1000 mL, pH 7.00-7.50; (3) Simplified inorganic phosphorus liquid medium: glucose 10.0 g, ammonium sulfate 2.0 g, tricalcium phosphate 5.0 g, tryptone 0.5 g, deionized water 1000 mL, pH 7.00-7.50; (4) Simplified organic phosphorus liquid medium: glucose 10.0 g, ammonium sulfate 2.0 g, calcium phytate 2.0 g, tryptone 0.5 g, deionized water 1000 mL, pH 7.00-7.50; (5) PDA culture medium for preservation: potato 200 g, glucose 20 g, agar 18 g, distilled water 1000 mL, pH natural; (6) LB medium (g / L): tryptone 10.0 g, sodium chloride 10.0 g, yeast extract 5.0 g. All the media were sterilized at 121℃ for 20 min.

[0045] 1.2 Effective phosphorus content determination method

[0046] 2 mL of each treatment was centrifuged at 10,000 rpm / min for 10 min, and the content of available phosphorus in the supernatant was determined according to the molybdenum-antimony anti-colorimetric method (Wen Chen. Improvement of molybdenum-antimony anti-colorimetric method for determination of total phosphorus in water [J]. Shanxi Chemical Industry, 2003) with a colorimetric cell of 10 mm light path and a wavelength of 700 nm, with no bacteria as a blank control. The absorbance value (OD) at a wavelength of 700 nm was determined as the ordinate, and the amount of dissolved phosphorus (x) was taken as the abscissa to draw a phosphorus standard curve. The linear regression equation of the molybdenum-antimony anti-phosphorus standard curve was y = 0.5031x + 0.0112, the correlation coefficient R = 0.9976, and the content of available phosphorus in the solution was calculated by the phosphorus standard curve regression equation to determine the phosphorus solubilizing ability of the strain. The phosphorus solubilizing rate was calculated according to the formula: phosphorus solubilizing rate (%) = (available phosphorus content of bacteria inoculation - available phosphorus content of control) / amount of added inorganic phosphorus source x 100. 700nm 2 The value of the absorbance at a wavelength of 700 nm (y) was taken as the ordinate, and the amount of dissolved phosphorus (x) was taken as the abscissa to draw a phosphorus standard curve. The linear regression equation of the molybdenum-antimony anti-phosphorus standard curve was y = 0.5031x + 0.0112, the correlation coefficient R = 0.9976, and the content of available phosphorus in the solution was calculated by the phosphorus standard curve regression equation to determine the phosphorus solubilizing ability of the strain. The phosphorus solubilizing rate was calculated according to the formula: phosphorus solubilizing rate (%) = (available phosphorus content of bacteria inoculation - available phosphorus content of control) / amount of added inorganic phosphorus source x 100.

[0047] 1.3 Screening of phosphorus-solubilizing fungus XZY3PSF

[0048] 1.3.1 Preliminary screening of phosphorus-solubilizing fungus XZY3PSF: 10 g of soil sample was weighed into 90 mL of sterile water, and a soil suspension was prepared by shaking at 28°C for 30 min. The soil suspension was gradient diluted to 10 -1 , 10 -2 After drying, the glass coating rod was used to coat the inorganic phosphorus solid medium, and the sealed film was inverted in a biochemical incubator at 28°C for culture. The transparent ring around the fungus colony on the plate was observed to preliminarily screen the phosphorus-solubilizing bacteria. The fungus colony with obvious transparent ring was picked and transferred to a PDA plate for purification culture, and served as the preliminary screening strain for re-screening of phosphorus-solubilizing fungus.

[0049] 1.3.2 Re-screening of phosphorus-solubilizing bacteria: 150 mL of inorganic phosphorus liquid medium was added to a triangular flask, and 200 μL of prepared spore suspension was inoculated. The culture was shaken at 28°C and 180 rpm / min for 5 days. The existing phosphorus-solubilizing control strains LGJPJ and X5 were inoculated with single colonies on LB liquid medium after shaking for about 16 h, and 200 μL of bacterial liquid was inoculated into inorganic phosphorus liquid medium and cultured at 37°C and 180 rpm / min for 5 days. 2 mL of each culture liquid was centrifuged to obtain the supernatant, and the content of available phosphorus in each culture liquid was determined according to the method to compare the content of available phosphorus in each culture liquid, and the high-efficiency phosphorus-solubilizing bacteria were screened.

[0050] ​1.3.3 Preliminary analysis of the mineralization ability of phosphorus-solubilizing bacteria to organic phosphorus: The phosphorus-solubilizing bacterial strain was picked and placed on a simplified version of the Monzinga solid medium. The mineralization ability of the strain to organic phosphorus was preliminarily analyzed by observing whether a transparent circle was formed around the fungal colony on the plate, measuring the colony diameter (d) and the phosphorus-solubilizing circle diameter (D), and calculating the soluble index (D / d). Meanwhile, 2 mL of 150 mmol / L pNPP was added to the medium plate to cover the phosphorus-solubilizing circle, and the color development result was observed after 4 hours. If yellow color was produced, it indicated that the phosphorus-solubilizing bacteria secreted phosphatase.

[0051] Screening results of phosphorus-solubilizing bacteria: On the inorganic phosphorus solid medium plate, a transparent circle was formed around the phosphorus-solubilizing strain. The strength of the phosphorus-solubilizing ability of the strain was preliminarily judged according to the size of the transparent circle diameter. The phosphorus-solubilizing ability of the strain obtained by preliminary screening was determined with existing phosphorus-solubilizing bacteria. After 5 days, the phosphorus-solubilizing ability reached 502.1 mg / L, which was 7.3 times and 9.4 times higher than that of LGJPJ (68.9 mg / L) and X5 strain (53.3 mg / L), respectively. The soluble index of the strain on the Monzinga solid medium plate reached 1.6 (A in the figure) on the 5th day, and yellow color was observed after adding pNPP (B in the figure), indicating that the phosphorus-solubilizing bacteria could secrete phosphatase. Figure 1 Figure 1

[0052] The screened phosphorus-solubilizing bacterial strain was named XZY3PSF, and further molecular identification and phosphorus-solubilizing property research were carried out.

[0053] 1.4 Identification of phosphorus-solubilizing fungus XZY3PSF

[0054] Extraction of DNA of phosphorus-solubilizing fungus XZY3PSF and determination of ITS and β-tubulin gene sequences:

[0055] (1) Extraction of DNA of phosphorus-solubilizing fungus XZY3PSF: The strain XZY3PSF purified by single spore was activated in PDA medium, and then fungal DNA was extracted according to the SDS method and stored at -20°C.

[0056] (2) ITS gene sequence amplification: PCR amplification was carried out with fungal universal primers ITS1 (5'-TCCGTAGGTGAACCTGCGC-3' (SEQ ID No. 1)) and ITS4 (5'-TCCTCCGCTTATTGATATGC-3' (SEQ ID No. 2)). The PCR amplification reaction system: template 1 μL, 10 mmol·L -1 ​​Primer 1 μL, Takara Mix 12.5 μL, supplemented with ddH2O to 25 μL system. PCR reaction conditions: 94℃ pre-denaturation 2 min, then 94℃ denaturation 30 s, 58℃ annealing 50 s, 72℃ extension 1 min for 35 cycles, finally 72℃ extension 7 min. The PCR amplification product was stored at 4℃.

[0057] (3) β-tubulin gene sequence amplification: PCR amplification was performed using fungal universal primers Bt2a (5'-GGTAACCAAATCGGTGCTGCTTTC-3' (SEQ ID No. 3)), Bt2b (5'-ACCCTCAGTGTAGTGACCCTTGGC-3' (SEQ ID No. 4)). PCR amplification reaction system: template 1 μL, 10 mmol·L -1 Primer 1 μL, Takara Mix 12.5 μL, supplemented with ddH2O to 25 μL system. PCR reaction conditions: 94℃ pre-denaturation 2 min, then 94℃ denaturation 30 s, 58℃ annealing 50 s, 72℃ extension 1 min for 35 cycles, finally 72℃ extension 7 min. The PCR amplification product was stored at 4℃.

[0058] (4) The product was detected by 1% agarose gel electrophoresis, and the PCR product was purified and sent to Shanghaigeng (Shanghai) Bioengineering Co., Ltd. for sequencing. The obtained sequence was compared and analyzed with all sequences in the database by Blast program in GenBank, and the phylogenetic tree was constructed by using DNAStar software.

[0059] It can be seen that the strain XZY3PSF was inoculated on PDA medium and cultured, the primary hypha of the strain XZY3PSF was white and transparent, green spores were produced after 3 days, gathered in the center, the surrounding hypha was white, and the back of the plate showed orange red. After 15 days of culture, the hypha almost covered the whole plate, the spores were gray green (A in the figure), the back of the plate was deep red (B in the figure). The sporogenous structure was 3 branches and whorled (C in the figure), the spores were oval, the size was 3.16-3.69 μm, and the spores were long chain (D in the figure). Figure 2 Figure 2 Figure 2 Figure 2 Figure 2

[0060] ​​​​​The results of homology comparison of the ITS gene sequence and the β-tubulin gene sequence of strain XZY3PSF showed that the ITS sequence of XZY3PSF had 100% homology with the ITS sequence of Talaromyces purpureogenus with GenBank accession number MN907642.1, and the β-tubulin sequence had 100% homology with the sequences of T. purpureogenus with GenBank accession numbers MK451197.1 and MK451193.1. The same strain with ITS and β-tubulin gene sequences was selected and the sequences were spliced, and a biological phylogenetic tree was constructed by using the adjacent method, and it was found that each accession number belonging to T. purpureogenus was in the same branch, and the similarity reached 99.8% (see Figure 3 ). According to the morphological observation and the comparison of ITS and β-tubulin gene sequences, XZY3PSF was identified as a new strain of T. purpureogenus.

[0061] 1.5 Analysis of the phosphate solubilizing ability of phosphate-solubilizing fungus XZY3PSF

[0062] 1.5.1 Preparation method of spore suspension

[0063] The mycelium of the strain was picked and placed in a PDA medium plate, which was incubated at 28°C until spores grew. The spores were washed with sterile water and filtered through sterile 3 layers of lens paper to prepare the spore suspension of the fungus. The spore count was about 7×10 6 spores / mL by microscopic examination.

[0064] 1.5.2 Analysis of the phosphate solubilizing ability of XZY3PSF strain in different concentrations of calcium phosphate

[0065] A 150 mL triangular flask was filled with 50 mL of inorganic phosphorus liquid medium without calcium phosphate, and 0.125 g, 0.250 g, 0.400 g, 0.500 g, 0.600 g, 0.750 g, and 1.000 g of calcium phosphate were added respectively to prepare inorganic phosphorus liquid medium with final concentrations of 2.5, 5.0, 8.0, 10.0, 12.0, 15.0, and 20.0 g / L respectively. 200 μL of prepared spore suspension was added, and the culture was incubated at 180 rpm / min and 28°C, then incubated at room temperature after 11 days. 2 mL of culture solution was taken at 1d, 3d, 5d, 7d, 9d, 11d, 13d, 15d, and 25d, and the effective phosphorus content was determined according to the effective phosphorus content determination method, and the pH value of the culture solution was also determined, with each treatment repeated 3 times.

[0066] Table 1 Difference comparison and analysis of the effective phosphorus content of XZY3PSF strain at 7d and 25d under different concentrations of calcium phosphate

[0067]

[0068] By Figure 4 As shown in Table 1, with the increase of the concentration of tricalcium phosphate, the highest available phosphorus content in the culture solution showed no significant change, and relatively speaking, the available phosphorus content in the culture solution with 5 g / L tricalcium phosphate was the highest, and the phosphorus solubilization rate (X value) with 2.5 g / L tricalcium phosphate was the highest, and the phosphorus solubilization rate (X value) decreased with the increase of the concentration of tricalcium phosphate; with the extension of the culture time, from the 13th day, the decreasing amplitude of the available phosphorus content in the supernatant showed an upward trend with the increase of the concentration of tricalcium phosphate, but was still extremely significantly lower than that in the supernatant on the 5th day or the 7th day, and on the 25th day, the available phosphorus content in the supernatant with 15 g / L and 20 g / L tricalcium phosphate was extremely significantly higher than that with other concentrations.

[0069] 1.5.3 Analysis of the phosphorus solubilizing ability of XZY3PSF strain under different spore suspension inoculation amounts

[0070] 150 mL triangular flask was filled with 50 mL of inorganic phosphorus liquid medium, and the prepared spore suspension was inoculated into the inorganic phosphorus liquid medium according to 0.2 μL, 2 μL, 20 μL, 200 μL and 2 mL, so that the final spore concentration was 28 spores / mL, 280 spores / mL, 2800 spores / mL, 28,000 spores / mL and 280,000 spores / mL respectively. The culture was incubated at 28°C on a shaking table at 180 rpm / min, and then incubated at room temperature after 11 days. 2 mL of culture solution was taken on the 1st day, 3rd day, 5th day, 7th day, 9th day, 11th day, 13th day, 15th day and 25th day, and the available phosphorus content was determined according to the available phosphorus content determination method, and each treatment was repeated for 3 times.

[0071] By Figure 5 It can be seen that with the increase of the five spore concentrations, the time of the highest soluble phosphorus content in the supernatant of each treatment was shortened, which was the 11th day, the 9th day, the 9th day, the 7th day and the 7th day respectively; with the extension of the culture time, the decreasing trend of the available phosphorus content in the supernatant of 28 spores / mL was relatively slow, while the rest of the spore concentrations showed extremely significant difference compared with the original highest peak value on the 25th day, especially the spore concentration of 280,000 spores / mL, which showed the most steep decreasing trend of the available phosphorus content, and the available phosphorus content on the 15th day was only 93.9 mg / L, and the available phosphorus content on the 25th day was only 10.3 mg / L, which was 5.6 times and 50.8 times different from that on the 7th day respectively.

[0072] 1.5.4 Analysis of the mineralization ability of XZY3PSF strain under different concentrations of calcium phytate

[0073] In the 150 mL triangular flask containing 50 mL (final concentration of calcium phytate 2.0 g / L, 5.0 g / L) of the simplified version of the Monkhina liquid medium, 200 μL of spore suspension was added, and the final concentration of spores was 2.8 × 104 spores / mL. After 11 days of culture at 180 rpm / min and 28°C, the culture was incubated at room temperature. 2 mL of culture solution was taken at 1d, 3d, 5d, 7d, 9d, 11d, 13d, 15d, 25d, 35d, and the effective phosphorus content was determined according to the effective phosphorus content determination method. Each treatment was repeated 3 times.

[0074] From Figure 6 It can be seen that in the test period, the effective phosphorus content in the culture solution with two concentrations of calcium phytate increases with the extension of the culture time, and the highest phosphorus solubilization efficiency is reached at 25d with a final concentration of 5.0 g / L calcium phytate. At the same time, with the increase of the concentration of calcium phytate, the effective phosphorus content in the culture solution also increases, and there is a very significant difference at different time points.

[0075] 1.6 Analysis of the antagonistic effect of phosphorus-solubilizing fungus XZY3PSF on 5 fungal pathogenic fungi

[0076] The antagonistic activity of phosphorus-solubilizing fungus XZY3PSF on banana wilt pathogen B2 strain, banana fruit rot pathogen Nbm-1 strain, cherry stem blight pathogen YSSGa-2 strain, YSSGa-10 strain, YSSGa-3 strain and Bacillus velezensis X05 on banana wilt pathogen B2 strain was determined by the method of plate confrontation. The information of fungal pathogens is shown in Table 2. The antagonistic activity detection method is described in the invention patent of Wang Jun et al. (Wang Jun, Huang Junsheng, Liang Changcong, Zhou You, Liu Lei, Yang Laying, Guo Likai. A method for determining the antagonistic activity of biocontrol bacterial metabolites under non-sterile conditions [P]. CN107937479A, 2018). A 0.5 cm fungal cake of each pathogen was inoculated in the center of the PDA solid culture medium plate. Two points 2.5 cm away from the center were selected on the cross-symmetric line, and a 0.5 cm fungal cake of phosphorus-solubilizing fungus XZY3PSF or 10.0 μL of LB shake culture X05 was inoculated. Each strain was repeated 3 times. The colony diameters were measured by cross method.

[0077] Inhibition rate = (colony diameter of control group - colony diameter of treatment group) / colony diameter of control group.

[0078] Table 2 Inhibition rate of phosphorus-solubilizing fungus XZYSPSF and Bacillus velezensis X5 strain on different plant pathogenic fungi

[0079]

[0080]

[0081] Results are shown in Figure 7 As shown in Table 2, the inhibition rate of the phosphate-solubilizing fungus XZY3PSF on Fusarium oxysporum f. sp. cubense, Pilobolus crystallinus and Pestalosphaeria sp. reached 66.2% to 77.0% among the five plant pathogenic fungi tested, and the inhibition rate of the phosphate-solubilizing fungus XZY3PSF on Fusarium oxysporum f. sp. cubense was not significantly different from the antagonistic efficiency of Bacillus velezensis X05 strain. Therefore, the phosphate-solubilizing fungus XZY3PSF has strong bacteriostatic ability and a broad spectrum of bacteriostatic spectrum.

[0082] The above only describes the preferred embodiments of the present application, and it should be noted that for those skilled in the art, without departing from the principles of the present application, a number of improvements and refinements can be made, and these improvements and refinements should also be considered as the protection scope of the present application.

Claims

1. A phosphate solubilizing fungus XZY3PSF, characterized in that, The taxonomic name of the phosphorus-solubilizing fungus XZY3PSF is Talaromyces purpureogenitus Talaromyces purpureogenus , the strain number is XZY3PSF, and the accession number is GDMCC No:62750.

2. The application of the phosphorus-solubilizing fungus XZY3PSF in soil phosphorus solubilization or fertilizer phosphorus solubilization according to claim 1.

3. Use according to claim 2, characterized in that, The spore suspension of the phosphorus-solubilizing fungus XZY3PSF is used for soil phosphorus solubilization or fertilizer phosphorus solubilization.

4. Use according to claim 3, characterized in that, The spore suspension has a spore count of 5 x 10 6 ~ 1 x 10 7 spores / mL.

5. Use according to claim 3, characterized in that, The preparation method of the spore suspension comprises the following steps: mycelium of the phosphorus-solubilizing fungus XZY3PSF is inoculated into culture medium, and the culture is incubated at constant temperature of 26-30℃ until spores grow, the spores are washed with sterile water, and filtration is performed to obtain the spore suspension.

6. Use according to claim 5, characterized in that, The culture medium comprises PDA culture medium comprising the following components in parts by weight: potato 190-210 parts, glucose 15-25 parts, agar 15-20 parts, and water 900-1100 parts.

7. The use of the phosphate-solubilizing fungus XZY3PSF according to claim 1 for inhibiting plant pathogenic fungi, characterized in that, The plant pathogenic fungus is Fusarium oxysporum f. sp. cubense Fusarium oxysporum f. sp. cubense , Mycosphaerella graminicola Gliomastix sp. or Pestalotiopsis microspora Pestalotiopsis sp. .

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