An endophytic fungus G22 in the roots of *Platycladus orientalis* and its application

Abstract

The present invention discloses an endophytic fungus G22 from the root of Fujian cypress and its application. The endophytic fungus G22 is classified and named as Trichoderma harzianum ( TrichodermaHarzianum ) G22, which was deposited in the General Microbiology Center of the China Microbiological Culture Collection Committee on October 13, 2023, with the deposit number CGMCC No.40845, and the deposit address is the Institute of Microbiology, Chinese Academy of Sciences, No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing. Through potted phosphorus solubilization test and growth promotion analysis, it was found that the endophytic fungus G22 can convert the insoluble phosphorus in the soil that is difficult for plants to absorb and utilize into soluble effective phosphorus that can be directly absorbed and utilized, which can better promote plant growth and increase the plant seedling height, ground diameter, total root length, root surface area and root volume.

Description

Technical Field

[0001] The invention belongs to the field of microorganisms, and particularly relates to an endophytic fungus G22 from the roots of Fujian cypress and an application thereof. Background Art

[0002] Phosphorus is a key link in the growth and development of plants. It participates in many physiological processes such as flower bud differentiation, energy metabolism, and genetic variation. It plays an important role in the accumulation of plant biomass and is also a limiting factor for plant growth. Most of the timber forests in the collective forest areas in southern my country are distributed in low mountains and hills. The soil under the forest is mostly acidic red soil with extremely low effective phosphorus content. 95% to 99% of phosphorus exists in the form of insoluble calcium, iron, aluminum compounds and phytic acid compounds. Existing studies have mostly considered this to be one of the main factors restricting the sustainable management of artificial forests in the collective forest areas in the south. The lack of available phosphorus in the soil has significantly changed the sustainable development capacity of the plant living environment. Research on improving the current situation of insufficient effective phosphorus in the red soil area in the south to support the growth and development of trees mainly focuses on the following aspects: application of phosphorus fertilizer, silicon fertilizer, and tree species symbiosis. Although the above measures have achieved some results, the utilization efficiency of conventional chemical fertilizers is low, and excessive fertilization can easily cause soil acidification and compaction, reduced soil buffering performance and ecological environmental damage. Unscientific tree species symbiosis will lead to uneven distribution of soil nutrients. Existing measures are still unable to effectively and cost-effectively improve the shortage of available phosphorus in the soil under the forest.

[0003] Related studies have pointed out that the interaction between beneficial microorganisms and trees can mediate the adaptation of trees to adversity and enhance their ability to cope with stress. Domestic and foreign scholars have screened out a variety of microorganisms from plants that have the ability to solubilize phosphorus and enhance plant stress resistance and disease resistance. In the field of agricultural production, the development and utilization of phosphorus-solubilizing microorganisms has been proven in practice to be an effective means to assist or replace conventional fertilizers to solve soil fertility decline and improve land productivity. In recent years, continuous reports of symbiotic phosphorus-solubilizing microorganisms in trees have also provided research ideas for alleviating the problem of phosphorus stress in the growth environment of trees. Therefore, trying to tap into the microbial resources of trees from the perspective of mutualistic symbiosis between microorganisms and trees and effectively improve the efficiency of phosphorus utilization by trees has important practical significance for the sustainable management of artificial forests. Summary of the invention

[0004] The purpose of the present invention is to provide an endophytic fungus G22 from the roots of Fujian cypress and its application.

[0005] To achieve the above object, the present invention adopts the following technical solution:

[0006] An endophytic fungus G22 from the roots of Fujian cypress, the strain is classified and named Trichoderma Harzianum G22, and was deposited in the General Microbiology Center of China Microorganism Culture Collection Administration on October 13, 2023, with the deposit number CGMCC No. 40845, and the deposit address is the Institute of Microbiology, Chinese Academy of Sciences, No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing.

[0007] The above-mentioned endophytic fungus G22 from the roots of Fujian cypress is used for degrading insoluble phosphates, wherein the insoluble phosphates are iron phosphates.

[0008] The above-mentioned endophytic fungus G22 in the roots of Fujian cypress is used to promote plant growth, and the promotion of plant growth includes increasing the plant's seedling height, ground diameter, total root length, root surface area and root volume. The plant is Fujian cypress.

[0009] A phosphate-solubilizing and growth-promoting biological fertilizer, comprising the above-mentioned endophytic fungus G22 from the roots of Fujian cypress.

[0010] The above-mentioned application of the phosphate-dissolving and growth-promoting biological fertilizer in dissolving insoluble phosphates in the soil and promoting plant growth is specifically to apply the phosphate-dissolving and growth-promoting biological fertilizer in the form of a bacterial suspension to the soil where the roots of the plants are located to cultivate the plants; the plant is Fujian cypress.

[0011] The significant advantages of the present invention are:

[0012] Endophytic fungus G22 can significantly increase the available phosphorus content in the soil, reduce the insoluble iron-phosphorus content in the soil, and significantly improve the seedling phenotype (seedling height, ground diameter), root morphology (total root length, root surface area, root volume), and soil phosphorus content (available phosphorus, insoluble phosphorus), thereby helping to improve the adaptability of seedlings to low-phosphorus stress environments. BRIEF DESCRIPTION OF THE DRAWINGS

[0013] Figure 1 is the soluble phosphorus content of different endophytic fungi in the insoluble iron-phosphorus liquid culture medium.

[0014] Figure 2 This is the colony morphology of the endophytic fungus G22.

[0015] Figure 3 It is the phosphate-solubilizing ability of endophytic fungus G22 in liquid culture medium containing poorly soluble iron and phosphate. DETAILED DESCRIPTION

[0016] In order to make the contents of the present invention easier to understand, the technical solution of the present invention is further described below in conjunction with specific implementation methods, but the present invention is not limited thereto.

[0017] Example 1: Isolation, screening and identification of phosphate-solubilizing endophytic fungi of Cypress

[0018] S1: Plant disinfection:

[0019] Select three disease-free and healthy Fujian cypress plants, and wash the surface soil and dust with ultrapure water. The root of each plant is sampled, and three replicates are taken from each root system. After the root sample is washed, wipe the surface moisture with sterile paper, cut a 1cm small section with a sterile scalpel, and disinfect it in a clean bench. First disinfect it with 75% ethanol for 30 seconds, then rinse it with sterile water twice, then disinfect it with 1% to 2% NaClO for 3 to 5 minutes, and finally rinse it repeatedly with sterile water for 3 to 5 times. After disinfection, use sterile paper to absorb the surface moisture of the root sample.

[0020] S2: Endophytic fungal culture:

[0021] The root sample disinfected by S1 was cut into 1.0 mm slices in a clean bench, and endophytic fungi were cultured by solid culture method and liquid culture method. The specific methods are as follows:

[0022] ① Solid culture method: Inoculate the sliced ​​root sample tissue on the sterilized PDA plate under a sterile environment, mark it and culture it in a 28℃ constant temperature incubator for 5 to 7 days. After the hyphae grow out, select the strain to isolate the strain.

[0023] ②Liquid culture method: inoculate the sliced ​​root sample tissue into PDB liquid culture medium, mark it, and culture it in a constant temperature shaker (28℃, 220r / min) for 3-5 days. After the culture is completed, aspirate the bacterial solution for dilution (10 -1 , 10 -2 , 10 -3 ) and spread it on the PDA plate, mark it and culture it in a constant temperature incubator at 28℃ for 5-7 days. After the hyphae grow out, select the strains to separate them.

[0024] S3: strain purification:

[0025] The strain grown in S2 was purified 2-3 times by streaking method. The purified strain was inoculated on slant medium and stored in a 4°C refrigerator.

[0026] S4: Screening of phosphate-solubilizing endophytic fungi:

[0027] Phosphate-solubilizing bacteria screening was performed by shaking flask rescreening. The isolated strains were inoculated into insoluble iron-phosphorus liquid culture medium, and the uninoculated strains were used as control treatment. The culture was cultured in a constant temperature shaker for 5-7 days (28°C, 220r / min), and 3 groups of replicates were set for each treatment. After the culture, the culture medium of the strain was centrifuged (4°C, 4000×g, 10min), and the soluble phosphorus absorbance of the bacterial solution was determined by molybdenum antimony colorimetry, and the soluble phosphorus content of the bacterial solution was calculated using the phosphorus standard curve. The uninoculated insoluble iron-phosphorus liquid culture medium was used as the control (CK). The formula of the insoluble iron-phosphorus liquid culture medium is: glucose 10g, (NH4)2SO4 0.1g, MgSO4·7H2O 0.25g, MgCl2 5.0g, KCl 0.02g, FePO4 5.0g, ultrapure water 1L, pH 7.0; 115°C high-pressure steam sterilization for 30min.

[0028] The results show that Figure 1 ), a total of 18 strains had the ability to increase phosphorus, and the soluble phosphorus content in the culture fluid of each strain ranged from 94.94 to 105.85 μg / mL, an increase of 0.79 to 11.69 μg / mL compared with CK; among them, strain G22 had the best phosphorus-increasing effect, with a soluble phosphorus content of 105.85 μg / mL in the culture fluid and an increased phosphorus amount of 11.69 μg / mL.

[0029] S5: Identification of phosphate-solubilizing endophytic fungi

[0030] At 28℃, strain G22 was inoculated on a PDA plate and cultured for 7 days. The colonies were white in the early stage (2-3 days), the hyphae were white and loose in the early stage, the colonies were flat in the center, the edges were raised, and they were all round. In the late stage (4-7 days), they were cyan and evenly distributed on the PDA plate from the center point. The surface was dry and developed into green in the late stage. Single colonies were scattered and opaque ( Figure 2 ).

[0031] The genomic DNA of strain G22 was extracted using a kit, and then fungal ITS universal primers were used. PCR amplification was performed using the Master Mix (Code No.: TSE003) system.

[0032] PCR amplification reaction system:

[0033]

[0034]

[0035] PCR reaction conditions:

[0036]

[0037] The PCR product was subjected to agarose gel electrophoresis, and then the gel block was placed in a gel imaging instrument to collect images. Under a UV transilluminator, the target band was cut with a scalpel. After continuous purification, 2 μl of the sample was added to 5 μl of 1.4× bromophenol blue and the mixture was spotted into 0.8% identification gel. The identification gel was placed in a gel imaging instrument to collect images. The gel images before and after purification were compared. The template concentration of each well was marked on the PCR record sheet according to the PCR quantitative standard and diluted to the specified concentration. The diluted template was water bathed for 5 minutes and centrifuged (4000 rpm).

[0038] The sequencing reaction system and cycling conditions are as follows:

[0039]

[0040] The obtained ITS sequence (SEQ ID NO.1) was submitted to the NCBI database for sequence comparison analysis, and the strain G22 was preliminarily identified as Trichoderma Harzianum.

[0041] S6: Phosphate solubilization ability of endophytic fungus G22

[0042] Strain G22 was activated in PDB and grown to OD 600 When the value reaches 0.6, it is inoculated into the insoluble iron-phosphorus liquid culture medium, and the non-inoculated treatment is set as the control group, and each treatment is set up with 3 replicates. From the beginning of culture, the bacterial liquid is taken for determination after 24h, 48h, 72h, 96h, and 120h, and the bacterial liquid is centrifuged (4℃, 4000×g) for 10min, and the supernatant is taken and vacuum filtered through a 0.22μm filter membrane for determination of soluble phosphorus content.

[0043] result( Figure 3 ) showed that in the insoluble iron-phosphorus liquid culture medium, under the action of the G22 strain, the soluble phosphorus content showed a trend of first rising and then falling. Within 12-72 hours, the soluble phosphorus content increased from 69.84μg / mL to 89.96μg / mL, and the soluble phosphorus content did not change much after 72 hours. The soluble phosphorus content of the uninoculated CK group remained basically unchanged, maintained between 61.47μg / mL and 66.54μg / mL.

[0044] Example 2: Application of phosphate-solubilizing endophytic fungi phosphate-solubilizing method

[0045] S1: Bacterial liquid preparation

[0046] First, the G22 strain was cultured in liquid (PDB) and when its OD 600 When the concentration reached 0.6, centrifugation was performed (4°C, 4000×g, 10 min), the supernatant medium was removed, and the cells were resuspended in sterile water to obtain G22 bacterial solution (concentration of 1×10 7spores / mL). The G22 strain was cultured in liquid (PDB) and when its OD 600 When the pH reached 0.6, the cells were inactivated (121°C high-pressure steam inactivation for 30 min) and then centrifuged (4°C, 4000×g, 10 min). The supernatant culture medium was removed and the cells were resuspended in sterile water of an equal volume to that of the G22 bacterial solution to obtain a control bacterial solution.

[0047] S2: Soil disinfection

[0048] Before planting, the red soil (Table 1) and sand were fumigated and disinfected. Dazomethane (45 g·m -2 ), sealed with black film. After 25 days, the film was removed for ventilation, and after 5 days of drying, deep plowing was used for growth promotion experiments.

[0049] Table 1 Nutrient content of tested soil

[0050]

[0051] S3: Planting

[0052] The healthy, half-year-old second-generation seed garden improved species of Fujian cypress were selected and potted in a mixture of red soil and sand (3:1 m / m) (15.5 cm in diameter, 12 cm in bottom diameter, 14 cm in height, with drainage holes at the bottom and good ventilation) in a constant temperature greenhouse (24 °C, 65% humidity, 1×10 4 lux, photoperiod 12h) for 1 month, during which weeds were removed regularly to prevent insect pests, and 100mL of sterile water was poured every 5 days.

[0053] S4: Inoculation

[0054] After the seedlings were grown, healthy plants with consistent growth and good growth conditions were selected for inoculation. There were 2 groups of treatments in total (the inoculation group used G22 bacterial solution, and the control group CK used control bacterial solution), with 10 replicates in each group. Each pot of plants was watered with 25 mL of bacteria (applied to the soil where the roots of the plants were located), for a total of 20 plants. The watering treatment was performed 4 times in total, and the inoculation was performed every 7 days. After the last inoculation, the inoculation time was recorded. The seedlings were harvested on the 30th and 60th days, respectively, and the seedling height, ground diameter, biomass, root data, and soil nutrients were measured.

[0055] S5: Index determination

[0056] The seedling height was measured with a tape measure and the ground diameter was measured with a vernier caliper. The aboveground (stem, leaf) and underground (root) parts of Fujian cypress were weighed freshly and then withered at 105℃ for 45min. The samples were then dried at 75℃ to constant weight, and their aboveground and underground biomass were calculated.

[0057] As shown in Table 2, one month after inoculation, the height growth and ground diameter growth of Fujian cypress seedlings treated with G22 strains were significantly different from those treated with CK (P<0.05). The height growth of Fujian cypress seedlings treated with G22 strains was 2.36 cm, and the ground diameter growth was 0.46 mm. Two months after inoculation, the height growth of Fujian cypress seedlings inoculated with G22 strains was significantly different from that of CK (P<0.05), which was 4.44 cm, and the ground diameter growth was 1.08 mm.

[0058] Table 2 Phenotypic data of Fujian cypress inoculated with G22 strain

[0059]

[0060] As shown in Table 3, one month after inoculation, the total root length of the seedlings of Fujian cypress treated with G22 strain was significantly different from that of CK (P<0.05), which was 86.73 cm; the root surface area of ​​the seedlings of Fujian cypress treated with G22 strain was also significantly different from that of CK, which was 242.12 cm 2 , while CK is 158.09cm 2 In terms of promoting the increase of root volume, the G22 strain treatment had a significant effect. The root volumes of the Fukienan cypress seedlings in the G22 strain treatment group and the CK treatment group were 56.17 cm 3 and 32.86cm 3 Two months after inoculation, the total root length, root surface area and root volume of the Fukienan cypress seedlings treated with G22 strain were significantly increased compared with those treated with CK.

[0061] Table 3 Data of the root system of Fujian cypress inoculated with G22 strain

[0062]

[0063] The soil samples around the roots of the plants were collected by the quartering method to determine the total phosphorus, available phosphorus and insoluble iron phosphorus.

[0064] As shown in Table 4, one month after inoculation with G22 strain, compared with CK, the available phosphorus content in the soil was 3.24 mg / kg, an increase of 25.10%; the insoluble iron phosphorus content in the soil was 23.27 mg / kg, a decrease of 4.31 mg / kg compared with CK. Two months after inoculation, the available phosphorus content in the soil under the treatment of G22 strain was 6.23 mg / kg, an increase of 59.34%; the insoluble iron phosphorus content was 7.47 mg / kg less than that of CK. It can be seen that inoculation with G22 strain can efficiently dissolve the insoluble iron phosphorus in the soil and increase the available phosphorus content in the soil.

[0065] Table 4 Phosphorus content in soil inoculated with G22 strain

[0066]

[0067] The above description is only a preferred embodiment of the present invention. All equivalent changes and modifications made according to the scope of the patent application of the present invention should fall within the scope of the present invention.

Claims

1. An endophytic fungus G22 from the roots of Fujian cypress, characterized by: The endophytic fungus G22 is classified and named as Trichoderma harzianum ( Trichoderma Harzianum ), which was deposited in the General Microbiology Center of China Microorganism Culture Collection on October 13, 2023, with the deposit number CGMCC No.40845, and the deposit address is the Institute of Microbiology, Chinese Academy of Sciences, No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing.

2. The use of the endophytic fungus G22 from the roots of Fujian cypress as claimed in claim 1 in degrading insoluble phosphates, characterized in that: The sparingly soluble phosphate is iron phosphate.

3. Use of the endophytic fungus G22 from the roots of Cupressus fujianensis as claimed in claim 1 in promoting plant growth.

4. The use according to claim 3, characterized in that: The promoting plant growth includes increasing the plant's seedling height, ground diameter, total root length, root surface area and root volume.

5. The use according to claim 3, characterized in that: The plant is Fujian Cupressus.

6. A phosphate-dissolving and growth-promoting biological fertilizer, characterized in that: The phosphate-solubilizing and growth-promoting biological fertilizer comprises the endophytic fungus G22 in the roots of the Fujian Cypress according to claim 1.

7. The use of the phosphate-dissolving and growth-promoting biological fertilizer according to claim 6 in dissolving sparingly soluble phosphates in soil and promoting plant growth, characterized in that: The sparingly soluble phosphate is iron phosphate.

8. The use according to claim 7, characterized in that: The promoting plant growth comprises increasing the plant's seedling height, ground diameter, total root length, root surface area and root volume, and the plant is Fujian cypress.

9. The use according to claim 7, characterized in that: The phosphate-dissolving and growth-promoting biological fertilizer is applied to the soil where the plant roots are located in the form of a bacterial suspension to cultivate the plants.

Images