Lamproporus strain cxl-18 and uses thereof

Abstract

The present application relates to the field of biotechnology, in particular to a strain CXL-18 of a layered near-melanosporum and application thereof.The strain CXL-18 of the present application can effectively promote the growth of pear trees, and also improve the drought resistance of pear trees; after the pear trees are inoculated with the strain, the plant height, stem diameter, leaf area and dry weight are all significantly higher than those of the control group without inoculation; from the appearance, the growth of the plants is obviously better than that of the control group; under the stress of moderate drought conditions, the leaf area is significantly increased by 33.50% compared with the control, and the relative water content of the leaf is significantly increased by 27.06% compared with the control; from the growth of the plants, the growth is also better than that of the control group, which shows that the strain CXL-18 has good growth-promoting ability for pear trees, can improve the drought resistance of pear trees, and can provide a new idea for the research and development of pear tree biological bacterial fertilizer.

Description

[Technical Field]

[0001] This invention relates to the field of biotechnology, and in particular to the CXL-18 strain of *Glomus latae* and its applications. [Background Technology]

[0002] Arbuscular mycorrhizal (AM) fungi are rhizosphere soil microorganisms that obligately form symbiotic relationships with plant roots. Approximately 80% of terrestrial plants can form mutualistic symbionts with AM fungi under natural conditions. Previous studies have shown that AM fungi have multiple functions in plant growth and development. AM fungi can expand the root absorption area and enhance absorption capacity by altering root architecture and the extension of extraroot hyphae, promoting the host plant's absorption and utilization of nutrients and water, changing plant growth status, and improving plant resistance to biotic and abiotic stresses. AM fungi are a good source of bio-fertilizers. However, actual research has revealed that there are many types of AM fungi, and different species vary greatly. Not all AM fungi can promote the growth of all plants; often, a certain AM fungus can specifically promote the growth of one or a few plants. Similarly, not all AM fungi have the ability to improve the plant's resistance to environmental stress. This means that if AM fungi are to be used as bio-fertilizers, it is necessary to continuously screen for superior strains, enrich the germplasm bank, and identify more strains with better agronomic characteristics. This is the foundational research for improving the application of AM fungi in the production of organic bio-fertilizers. [Summary of the Invention]

[0003] In view of the above, screening for more AM fungi that have good growth-promoting effects on plants and improve their resistance to environmental stress is the basic research for the application of AM fungi in biofertilizers. The purpose of this invention is to provide a new AM fungus, strain Claroideoglomus lamellosum CXL-18, which promotes the growth of pear rootstock and improves the drought resistance of pear trees.

[0004] To achieve the above objectives, this invention screened a strain of arbuscular mycorrhizal fungus (Claroideoglomus lamellosum) CXL-18, with the accession number CGMCC No. 40331. It belongs to the kingdom Fungi, genus Claroideoglomus, species lamellosum. This arbuscular mycorrhizal fungus is deposited at the China General Microbiological Culture Collection Center, address: Institute of Microbiology, Chinese Academy of Sciences, No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, on October 17, 2022.

[0005] The strain used in this application was obtained from the rhizosphere soil of the 'Chuxia Lv' pear tree in Liyuan, Lishui District, Nanjing City, Jiangsu Province, with Pyrus pyrifolia as the rootstock.

[0006] The present invention also includes a fungal agent comprising the Claroideoglomus lamellosum strain CXL-18.

[0007] The present invention also includes the application of the Claroideoglomus lamellosum strain CXL-18 or the inoculum agent in promoting the growth of pear trees.

[0008] The present invention also includes the application of the Claroideoglomus lamellosum strain CXL-18 or the inoculum agent in improving the drought resistance of pear trees.

[0009] Furthermore, the pear tree mentioned is Pyrus pyrifolia.

[0010] The present invention also includes a method for promoting pear tree growth or improving the drought resistance of pear trees by using the Claroideoglomus lamellosum strain CXL-18. The method is as follows: CXL-18 strain is prepared by inoculating corn or clover host to obtain CXL-18 inoculum, and then CXL-18 inoculum is directly inoculated into the propagation substrate of pear seedlings for propagation of pear seedlings.

[0011] The present invention also includes a method for promoting pear tree growth or improving the drought resistance of pear trees by applying the Claroideoglomus lamellosum strain CXL-18, wherein the method is as follows: pear tree seedlings are directly transplanted into a cultivation substrate containing CXL-18 inoculant.

[0012] The present invention has the following beneficial effects:

[0013] The novel strain of this application, after morphological and molecular biological identification, was identified as belonging to the genus *Claroideoglomus*, species *lamellosum*. Tests showed that strain CXL-18 effectively promoted pear tree growth and improved the pear tree's resistance to drought. After inoculation with this strain, the pear trees exhibited significantly higher plant height, stem diameter, leaf area, and dry weight than the uninoculated control group, and their growth was noticeably superior to the control group. Under moderate drought stress, the leaf area increased significantly by 33.50% compared to the control, and the relative water content of the leaves increased significantly by 27.06%. The plant growth was also superior to the control group. This demonstrates that strain CXL-18 has excellent growth-promoting ability for pear trees and can improve their drought resistance. This strain can provide new insights for the research and development of bio-fertilizers for pear trees. [Attached Image Description]

[0014] Figure 1 The images show the morphological characteristics of strain CXL-18 of this invention as captured by a microscope. In the images, A shows the morphology of spores and sporangia in sterile water with a floater; B shows the morphology of spores and detachment marks in PVLG floater; C shows the morphology of the spore wall structure and detachment marks in Melzer's + PVLG floater; and D shows the spores and staining reaction in lactic acid cotton blue reagent.

[0015] Figure 2 This is a phylogenetic tree diagram of the CXL-18 strain of the present invention;

[0016] Figure 3 The figures show the experimental results of the CXL-18 strain of the present invention on potted pear seedlings; where Figure A is the blank control (CK) and Figure B is the treatment group inoculated with CXL-18 inoculum.

[0017] Figure 4 The figures show the experimental results of different AM fungal agents of this invention on potted pear seedlings under moderate drought conditions; the left figure shows the growth of potted plants in each treatment group under normal conditions, and the right figure shows the growth of potted plants in each treatment group under moderate drought conditions.

Detailed Implementation Methods

[0018] The present invention will be further described below with reference to the accompanying drawings, embodiments, and experiments.

[0019] Example 1:

[0020] This invention screened out a strain of *Claroideoglomus lamellosum*, CXL-18. It belongs to the kingdom Fungi, genus *Claroideoglomus*, species *lamellosum*. Strain CXL-18 is deposited at the China General Microbiological Culture Collection Center (CGMCC), located at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences, with accession number CGMCC No. 40331, and deposited on October 17, 2022.

[0021] The CXL-18 strain was classified and identified as follows:

[0022] 1. Morphological classification of strains

[0023] like Figure 1The image shown is a microscopic photograph of the morphological characteristics of this fungus. The image reveals that the spores of CXL-18 are solitary, yellowish-brown, spherical or nearly spherical, 80.15-125.33 μm, with an average of 99.88 μm (n = 106). The spore wall consists of three layers. L1 is a transparent to pale yellow lamellar wall, <1 μm, unreacted in Melzer's reagent, but stained light blue in cotton blue, and is often easily degraded. L2 is a lamellar wall, pale yellow to yellow, 3.58-6.3 μm, composed of multiple sublayers, and unreacted. L3 is a membranous wall, <1 μm, pale pink in Melzer's reagent, and stained light blue to blue in cotton blue. The mycelium is transparent to pale yellow, and its wall is connected to the spore walls L1 and L2.

[0024] 2. Molecular biological identification

[0025] The first PCR amplification was performed using the universal primers Geo11 / GeoA2 for fungi, followed by a second amplification using the currently universal primers NS31 / AML2 for AM fungi. The PCR products were sequenced to obtain the SSU sequence. A phylogenetic tree was constructed using highly similar sequences downloaded from the NCBI website. The results are as follows: Figure 2 As shown, it is highly similar to the genus Claroideoglomus, and most closely related to the species lamellosum of the genus Claroideoglomus.

[0026] Example 2:

[0027] Its effect on promoting the growth of pear seedlings:

[0028] I. Preparation of different AM fungal inoculants:

[0029] Using corn or clover as hosts, spores of strains CXL-18, Fm, or Aca, obtained through isolation, purification, and propagation, were inoculated into the roots of corn or clover seedlings. The seedlings were then planted in a culture medium sterilized at 121°C for 2 hours using a mixture of river sand and zeolite in a 3:1 volume ratio. 50–100 CXL-18 spores were inoculated into the roots of one corn or 20 clover plants. The plants were cultured under light for 14 weeks, with 200 mL of a 1 / 2 concentration Hoagland nutrient solution applied every two weeks during this period. After removing the stems from the 14-week-old corn or clover plants, the mycelia and spores of *Gastromycium lamellae* (lamellae of *Gastromycium lamellae*) in the roots and culture medium were retained, thus obtaining several different AM fungal inoculants, including CXL-18, Fm, and Aca.

[0030] Among them, Fm is a commercial strain with the Latin name Funneliformis mosseae; it was purchased from the Institute of Plant Nutrition and Resource Environment, Beijing Academy of Agricultural and Forestry Sciences, with the code BGC HUN03B.

[0031] Aca was obtained by the applicant from the rhizosphere soil of nectarine trees in Lishui District, Nanjing City, Jiangsu Province. The rootstock was Prunus persica, and its Latin name was identified as Acaulospora mellea.

[0032] II. Pot experiment on the growth-promoting effect of different AM fungi on pear seedlings:

[0033] Pear rootstock variety: Pyrus pyrifolia

[0034] Pear seeds were soaked in warm water, disinfected with sodium hypochlorite for 10 minutes, rinsed thoroughly with deionized water, wrapped in gauze, and placed in petri dishes at 4 degrees Celsius. They were rinsed with water daily, and germination occurred after approximately two weeks. Seeds were sown in substrate trays, and after two weeks of growth, uniformly growing seedlings were transplanted. Each tray contained 600g of substrate (soil:sand = 1:1, with 2% organic fertilizer added), and different treatment groups were established, with eight replicates per group. Cultivation was carried out using conventional methods, with 50ml of 1 / 2 Hoagland nutrient solution applied every two weeks. Harvesting was conducted after 15 weeks of cultivation. Plant height, stem diameter, number of leaves, leaf area, chlorophyll content, and biomass were measured for each treatment group, along with root infection rate.

[0035] The above processing groups are as follows:

[0036] CXL-18 treatment group: Add CXL-18 inoculant at a dosage of 12g per pot;

[0037] Fm treatment group: Add Fm inoculant at a dosage of 12g per pot;

[0038] Aca treatment group: Add Aca inoculant at a dosage of 12g per pot;

[0039] Group CK: 12g of the three mixed bacterial agents were added to the filtrate per pot. The filtrate was obtained by filtering the bacterial agents through a 400-mesh sieve (no spores were found in the filtrate after passing through the 400-mesh sieve).

[0040] The results are shown in Tables 1 and 2:

[0041] Table 1. Effects of different inoculation treatments on pear seedling growth.

[0042]

[0043]

[0044] Table 1 shows that, compared with the control group, the pear seedlings inoculated with strain CXL-18 had significantly higher plant height, stem diameter, leaf area, and dry weight, while there were no significant differences in leaf number and chlorophyll content. The plant height, leaf area, and dry weight of the CXL-18 group were also significantly higher than those of the Fm and Aca groups, and its stem diameter was significantly higher than that of the Fm group, while there were no significant differences in leaf number and chlorophyll content. The overall effect was CXL-18 group > Aca group > CK group > Fm group. This indicates that strain CXL-18 has a good growth-promoting effect on pear trees, with the best effect, while strain Fm has no growth-promoting effect on pear trees.

[0045] Table 2. Infection status of pear seedlings under different inoculation treatments.

[0046] CXL-18 100 20.6 20.6 5.35 25.97 0.1 Fm group 80 16.15 20.19 6.01 37.18 0.6 Aca Group 90 30.15 33.5 9.19 30.48 0.45

[0047] Table 2 shows that different strains infected the roots of *Pyrus pyrifolia*. The CXL-18 group achieved a 100% infection rate, with an infection intensity and relative infection intensity of 20.6%, a branch abundance of 5.35%, a relative branch abundance of 25.97%, and a vesicle abundance of 0.1%. The branch abundance and vesicle abundance of the Fm and Aca groups were higher than those of the CXL-18 group. The infection rate was: CXL-18 group > Aca group > Fm group; the infection intensity was: Aca group > CXL-18 group > Fm group. This indicates that different inoculated strains had high infection effects on *Pyrus pyrifolia* seedlings, but the infection rates of the Aca strain isolated from peach trees and the CXL-18 strain isolated from pear trees were higher than those of the commercially available Fm strain.

[0048] The growth effect of pear tree bonsai experiment is shown in the figure. Figure 3 As shown in the figure, A is the blank control planted with wild pear, and B is the wild pear inoculated with CXL-18 inoculant. It is clearly visible in the figure that after inoculation with CXL-18 inoculant, the growth of the potted wild pear is significantly better than that of the control group, which shows that strain CXL-18 has a good growth-promoting effect on pear trees.

[0049] Example 3:

[0050] Its effect on drought resistance in pear seedlings:

[0051] I. Preparation of different AM fungal inoculants: The preparation method is the same as in Example 2.

[0052] II. Drought resistance test of pear seedlings inoculated with different AM fungi under drought conditions:

[0053] The seedling raising method was the same as in Example 2, with different treatment groups and 8 replicates per group. The seedlings were managed according to conventional cultivation methods, with 50 mL of 1 / 2 Hoagland nutrient solution applied every two weeks. After 9 weeks of normal growth following transplanting, all four groups of potted plants were subjected to moderate drought (40% of maximum soil water holding capacity), and harvested after 6 weeks of drought. Plant height, stem diameter, number of leaves, leaf area, chlorophyll content, biomass, and relative leaf water content were measured for each treatment group. Root segments were also taken to detect root infection rate, and mycelial density was measured using soil samples from the root zone.

[0054] The above processing groups are as follows:

[0055] CXL-18 treatment group: Add CXL-18 inoculant at a dosage of 12g per pot;

[0056] Fm treatment group: Add Fm inoculant at a dosage of 12g per pot;

[0057] Aca treatment group: Add Aca inoculant at a dosage of 12g per pot;

[0058] Group CK: 12g of the three mixed bacterial agents were added to the filtrate per pot. The filtrate was obtained by filtering the bacterial agents through a 400-mesh sieve (no spores were found in the filtrate after passing through the 400-mesh sieve).

[0059] The results are shown in Tables 3 and 4:

[0060] Table 3. Effects of different inoculation treatments on pear seedling growth under drought conditions.

[0061]

[0062] Table 3 shows that, under drought conditions, the treatment group inoculated with strain CXL-18 significantly increased the plant height and dry weight of pear seedlings compared with the groups inoculated with Fm and Aca, as well as the uninoculated control group. The stem diameter of the CXL-18 group was significantly higher than that of the Aca group, and its chlorophyll content was significantly higher than that of the Fm group, while the number of leaves showed no significant difference. The leaf area of ​​the CXL-18 group was significantly higher than that of the other three treatments, increasing by 32.14% compared to the control. Under drought conditions, the relative water content of leaves in the CXL-18, Fm, and Aca groups inoculated with AM fungi was significantly higher than that of the blank control.

[0063] Table 4. Infection status of pear seedlings under drought and normal conditions by inoculation with different AM fungi.

[0064]

[0065] Table 4 shows that under drought conditions, the infection rate and intensity of pear seedlings in the CXL-18 and Fm groups were higher than those in the normal water treatment, while those in the Aca group were relatively lower. The abundance of tufts in the CXL-18 group increased by 192.52%, which is beneficial for nutrient exchange between pear seedlings and mycorrhizae, and enhances the dependence of pear seedlings on mycorrhizal fungi. The abundance of vesicles in both the CXL-18 and Aca groups was significantly increased under drought conditions. Under drought conditions, the mycelial density of all inoculated treatments was significantly higher than that in the normal water treatment, increasing by 74.90%, 118.97%, and 36.83% in the CXL-18, Fm, and Aca treatments, respectively. This expanded the root system's absorption range, improved water absorption, and thus enhanced the drought resistance of pear seedlings.

[0066] The growth effect of pear trees in pots under drought conditions is shown in the figure. Figure 4 As shown, the left figure shows the growth of potted plants in each treatment group under normal conditions, and the right figure shows the growth of potted plants in each treatment group under moderate drought conditions. It is clear from the figures that under moderate drought conditions, the growth of pear potted plants in the treatment group inoculated with CXL-18 fungal agent is significantly better than that in the treatment group inoculated with Fm fungal agent, Aca fungal agent, and the control group of CXL-18 fungal agent filtrate. This indicates that strain CXL-18 still has a good growth-promoting effect on pear trees under moderate drought conditions and can significantly improve the drought resistance of pear trees compared with other AM fungi.

[0067] In summary, the strain CXL-18 of this application belongs to the genus Claroideoglomus and species lamellosum. It has a good growth-promoting effect on pear trees, and under moderate drought conditions, pear trees inoculated with this strain also showed good drought resistance, indicating that this strain is an arbuscular mycorrhizal fungus with good growth-promoting and drought-resistant functions.

[0068] The above description is a detailed description of the preferred embodiments of the present invention. However, the embodiments are not intended to limit the scope of the patent application of the present invention. All equivalent changes or modifications made under the technical spirit of the present invention should fall within the patent scope covered by the present invention.

Claims

1. Lamella near-luminiferous globosum ( Claroideoglomus lamellosum The application of strain CXL-18 in promoting pear tree growth and / or improving the drought resistance of pear trees is characterized by, The preservation number of the strain CXL-18 is CGMCCNO.40331.

2. A layered, nearly transparent globosum mold ( Claroideoglomus lamellosum A method for promoting pear tree growth or improving the drought resistance of pear trees using strain CXL-18, characterized in that... The method is as follows: CXL-18 bacterial agent is prepared by inoculating strain CXL-18 into corn or clover host, and then CXL-18 bacterial agent is directly inoculated into the propagation substrate of pear seedlings for propagation of pear seedlings. The preservation number of strain CXL-18 is CGMCC NO.40331.

3. A layered, nearly transparent globosum mold ( Claroideoglomus lamellosum A method for promoting pear tree growth or improving the drought resistance of pear trees using strain CXL-18, characterized in that... The method is as follows: pear seedlings are directly transplanted into a cultivation substrate containing CXL-18 bacterial agent, the preservation number of which is CGMCC NO.40331.

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