Crocus sativus rhizosphere fungus and application thereof

Abstract

The invention discloses saffron rhizosphere fungi and application thereof, and belongs to the technical field of plant rhizosphere microorganism growth promotion. The rhizosphere fungus is separated from rhizosphere soil of crocus sativus which is crocus sativus of orris family, belongs to ascomycophyta, Hamgutinomycetes and Mycomycota, is classified and named as Cadophora malum SRF536, and has the preservation number of CGMCC (China General Microbiological Culture Collection Center) No.41085. The invention further discloses a preparation method of the rhizosphere fungus. The rhizosphere fungus can promote lateral root growth and leaf growth of model plant arabidopsis thaliana, and increase the biomass of arabidopsis thaliana by 219.2%. The rhizosphere fungus provided by the invention has an obvious effect of promoting the growth of arabidopsis thaliana, and provides a microbial strain resource for research in the field of plant growth promotion.

Description

Technical Field

[0001] The invention relates to the technical field of plant growth promotion, in particular to a saffron rhizosphere fungus and an application thereof in promoting the growth of a model plant Arabidopsis thaliana. Background Art

[0002] Saffron is the dried stigma of Crocus sativus L., a plant of the genus Crocus in the family Iridaceae. Only the three-branched red filamentous stigma of the pistil can be used as medicine. It has low yield and high value. It is known as "plant gold" and has the effects of promoting blood circulation and removing blood stasis, cooling blood and detoxifying, relieving depression and calming the nerves. The main medicinal ingredients in saffron are crocin, crocetin, and crocin. Studies have found that saffron has the effects of improving cardiovascular and cerebrovascular function, calming the nerves, anti-tumor, preventing and treating Alzheimer's disease, relieving anxiety and depression, and treating diabetes.

[0003] At present, the actual production of saffron mostly adopts the "two-stage" cultivation method, in which the bulbs are placed indoors for half a year to cultivate and bloom, and then the flowers are picked indoors and planted outdoors in the field for half a year to reproduce new bulbs. This cultivation model has good saffron stigma quality, but the reproduction rate is low, which is not conducive to large-scale cultivation of saffron. At present, saffron cultivation has problems such as low bulb reproduction rate, non-standard cultivation technology, and increased incidence of diseases and insect pests. Relevant researchers have proposed solutions such as building a germplasm resource platform, scientifically planning planting, and comprehensively developing saffron by-products. However, the relevant scientific and technological advances to increase saffron production have not been clearly proposed.

[0004] Plant rhizosphere fungi refer to a class of fungi that can promote plant growth directly or indirectly in the soil around plant roots or attached to the root surface. They are one of the main sources of many plant growth inducers. Plant rhizosphere growth-promoting fungi have become an eco-friendly way to increase crop yields by increasing seed germination rate, promoting rhizome growth, and increasing chlorophyll production. Its mode of action includes the solubilization and mineralization of basic trace and macronutrients required by plants to regulate the balance of various plant growth processes. Plant rhizosphere fungi can also play a biological control role by inducing plant systemic resistance, thereby increasing plant yield. A large number of studies have shown that biological fertilizers prepared by rhizosphere fungi can promote plant growth. For example, Trichoderma harzianum biological fertilizers can not only improve the stem length, stem diameter, root diameter and other trait parameters and the yield of Bupleurum chinense, but also significantly increase the content of saikosaponins a, c and d. Inoculation with Funneliformis mosseae can effectively improve the fertility of the rhizosphere soil of Lycoris radiata, increase the accumulation of N, P, and K in different organs of the plant, and promote plant growth.

[0005] However, there are no reports on the application of saffron rhizospheric fungi to promote the growth of Arabidopsis thaliana. Summary of the invention

[0006] The purpose of the present invention is to provide a saffron rhizosphere fungus and its application in promoting the growth of Arabidopsis thaliana in view of the deficiencies of the prior art.

[0007] In the first aspect of the present invention, a plant rhizosphere fungus is provided, belonging to the Ascomycota, the Hammerglossum class, the Mollicutes order, and is classified and named as Cadophora malorum SRF536. The rhizosphere fungus is isolated from the rhizosphere soil of Crocus sativus L., a plant of the genus Crocus in the family Iridaceae, and is identified as Cadophora malorum SRF536 by microbial taxonomy. The strain has been preserved, the strain preservation number is CGMCC No. 41085, the preservation date is January 23, 2024, and the preservation unit is the General Microbiological Center (CGMCC) of the China Microbiological Culture Collection Administration, and the address is No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, and the zip code is 100101.

[0008] The solid culture characteristics of rhizosphere fungi described in the present invention are:

[0009] Incubated in the dark at 28℃ on potato dextrose agar (PDA), the mycelium grew slowly. The colony was round in shape, with a yellow-white surface, white fluffy mycelium in the center, yellow in the middle, and white mycelium visible on the outer layer. The inner layer of the back of the colony was yellow, and the outer layer was white. Figure 1 .

[0010] The morphological characteristics of the rhizosphere fungi under the microscope are: the mycelium is transparent, thin-walled, twisted, the hyphae are thin, branched, the surface is mostly smooth, and transparent spores are sparsely visible. Figure 2 .

[0011] The ITS amplification electrophoresis diagram of the rhizosphere fungi of the present invention is shown in Figure 3 , the ITS molecular sequence is shown in SEQ ID NO.1.

[0012] The sequencing results were compared on the NCBI website (http: / / blast.ncbi.nlm.nih.gov / Blast.cgi), and the sequence alignment similarity reached 99.81% with Cadophora malorum (MK626762), Cadophora malorum (MT666165), and Cadophora malorum (MT666152) on the same branch. MEGA7.0 was used for multiple sequence alignment, and the neighbor-joining (NJ) method was used for analysis. One sequence was randomly selected and the alignment was repeated 1000 times. The phylogenetic tree of the endophytic fungi of the present invention is shown in Figure 4 .

[0013] The second aspect of the present invention provides the use of the above-mentioned rhizosphere fungi in promoting the growth of Arabidopsis thaliana.

[0014] The above application, in which the fungus is beaten into a fungus cake and inoculated into the Arabidopsis growth medium, can promote the growth of the lateral roots and leaves of the model plant Arabidopsis and increase the biomass of Arabidopsis.

[0015] The above applications are mainly realized through the following technical solutions:

[0016] (1) Take SRF536 and pick up a small amount of mycelium with an inoculation needle under sterile conditions, inoculate it into sterilized PDA medium (the formula is 200 g potato, 20 g glucose, 15 g agar, 1000 mL distilled water, natural pH), and culture it in the dark at 28°C and 60% humidity for 5 days.

[0017] (2) Draw a horizontal line about 7 cm wide on the top of the 1 / 2 MS medium, and select Arabidopsis seedlings with similar growth conditions and place them at the line, with the spacing between the seedlings being 0.5 to 1.0 cm.

[0018] (3) Under sterile conditions, use a sterilized cork puncher with a diameter of 5 mm to punch a bacterial cake at the edge of the colony. Use sterilized tweezers to pick up the bacterial cake and place it 3 cm away from the horizontal line. Use the cork puncher to make a PDA block of the same size as the control group. Set up 3 replicates for each fungus.

[0019] (4) The plants were cultured in a 24°C light incubator with 16 h of light and 8 h of darkness. After 14 days, the biomass of the plants was measured and the root and leaf morphology of the Arabidopsis thaliana was observed under a microscope. It was clearly observed that SRF536 promoted the growth of Arabidopsis thaliana.

[0020] The present invention has the advantages that:

[0021] The saffron rhizosphere fungus Cadophora malorum SRF536 (Cadophora malorum) of the present invention can promote the growth of lateral roots and leaves of Arabidopsis thaliana after co-cultivation with Arabidopsis thaliana, increase the biomass of Arabidopsis thaliana, and increase the biomass by 219.2%. The rhizosphere fungus of the present invention has an obvious effect of promoting the growth of Arabidopsis thaliana, and brings broad application prospects in the fields of plant growth promotion and crop yield improvement. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] The present invention is further described below in conjunction with the accompanying drawings and embodiments;

[0023] Figure 1 :The colony morphology of Cadophora malorum SRF536 on PDA solid medium;

[0024] Figure 2 :Mycelial morphology of Cadophora malorum SRF536 under optical microscope;

[0025] Figure 3 :Gel electrophoresis of ITS sequence of Cadophora malorum SRF536 amplified by ITS5 and ITS4 primers;

[0026] Figure 4 : Phylogenetic tree of Cadophora malorum SRF536 based on ITS (NJ method);

[0027] Figure 5 :Photos and biomass statistics of the effect of Cadophora malorum SRF536 on the biomass of Arabidopsis thaliana;

[0028] Figure 6 :Microscopic photos showing that Cadophora malorum SRF536 promotes the growth of lateral roots and leaves of Arabidopsis thaliana. DETAILED DESCRIPTION

[0029] The rhizosphere growth-promoting fungus of the present invention is a strain separated from the rhizosphere soil of saffron in Chengcheng County, Weinan City, Shaanxi Province.

[0030] Embodiment 1:

[0031] The rhizosphere growth-promoting fungi were isolated by the following steps: 10 g of soil sample was weighed to prepare 1×10 -1 Concentration soil suspension. Take 1mL l×10 -1 The concentration of suspension was 1×10 -2 The soil suspension was diluted to 1×10 -3 , 1×10 -4 , 1×10 -5 100 μL of soil suspension was evenly coated on the PDA plate, and 3 replicates were set for each concentration. The plates were cultured in a fungal incubator for 5 days. Mycelia of colonies of different shapes and colors were picked on the plates, transferred to new culture media for purification and culture. After the culture was completed, the plates were observed and screened again to select suitable colonies. Mycelia at the edge of the colonies were inoculated on new plates. The screening, picking and culture were repeated to purify the strains. Finally, the endophytic fungus strain SRF536 of the present invention was isolated. Figure 1 As shown, the hyphae grow slowly, the colony is round in shape, with a yellow-white surface, white fluffy hyphae in the center, yellow in the middle layer, and white hyphae in the outer layer; the inner layer of the back of the colony is yellow and the outer layer is white. Figure 2) The mycelium is transparent, thin-walled, twisted, thin and branched, with a smooth surface and sparse transparent spores.

[0032] Take the frozen tube of SRF536 strain, pick a small amount of hyphae with an inoculation needle under sterile conditions and place it in a freshly prepared PDA medium for activation, and culture it at 28℃ in the dark until mature, for ITS sequence amplification and molecular identification. Extract the strain DNA according to the modified CTAB method, and use primers ITS5 and ITS4 to amplify its ITS sequence by PCR. PCR reaction cycle parameters amplification steps: 1, 95℃, 3min, initial denaturation; 2, 94℃, 40s, denaturation; 3, 52℃, 50s, annealing; 4, 72℃, 1min, extension; 5, 2-4 repeated cycles 35 times; 6, 72℃, 10min, extension. The PCR product detected by agarose gel electrophoresis was sent to Shanghai Shenggong Company for sequence determination, and the sequence is shown in SEQ ID NO.1. The sequence obtained from the PCR product was used as the target sequence, and the homologous sequence was searched in the GenBank database in NCBI. The reference sequence most similar to the morphological sequence was downloaded, and the neighbor-joining method (NJ) was used for phylogenetic analysis to determine the phylogenetic status of the strain to be identified, such as Figure 3 , 4 As shown. The rhizosphere growth-promoting fungal strain SRF536 of the present invention is on the same branch as Cadophora malorum (MK626762), Cadophora malorum (MT666165), and Cadophora malorum (MT666152), and the sequence alignment similarity reaches 99.81%. The fungus SRF536 (Cadophora malorum) belongs to the Ascomycetes, the Hammerglossum, the Mollicutes, and the genus Cadophora, and the strain collection number is CGMCC No.41085.

[0033] Embodiment 2:

[0034] Take the frozen tube of SRF536 strain, pick a small amount of mycelium with an inoculation needle under sterile conditions and place it in the newly prepared PDA medium for activation, and culture it at 28℃ in the dark until maturity; sterilize the Arabidopsis seeds in the clean bench using the three-step disinfection method, mix the seeds with sterilized agar water, and evenly sow them on 1 / 2MS medium. After placing it in a 4℃ refrigerator for 3 days, transfer it to a light incubator at 24℃ for culture, with 16h light and 8h dark.

[0035] Draw a horizontal line at a width of about 7 cm on the upper part of the 1 / 2MS culture medium, select 5 Arabidopsis seedlings with similar growth conditions and pick them up to the line to ensure that Arabidopsis grows on the same horizontal line, and the interval between the seedlings is 0.5-1.0 cm. Under sterile conditions, use a sterilized puncher with a diameter of 5 mm to punch a bacterial cake at the edge of the colony, use sterilized tweezers to clamp the bacterial cake and place it 3 cm away from the horizontal line, and use a puncher to make PDA blocks of the same size as the control group, with 3 replicates per group. Cultivate in a 24℃ light incubator with 16h light and 8h dark. After 14 days, measure the plant biomass and observe the roots and leaves of Arabidopsis under a microscope. The percentage of biomass increase is calculated according to the formula: increased biomass = (biomass of Arabidopsis in the treatment group - biomass of Arabidopsis in the control group) / biomass of Arabidopsis in the control group × 100%. The results showed that SRF536 significantly promoted the growth of Arabidopsis. Compared with the control group, the Arabidopsis in the SRF536-treated group had well-developed lateral roots, more hairy leaves, better growth potential, and a 219.2% increase in biomass. Figure 5 , 6 shown.

[0036] The preferred embodiments of the present invention have been specifically described above, but the present invention is not limited to the embodiments. Those skilled in the art may make various equivalent modifications or substitutions without violating the spirit of the present invention. These equivalent modifications or substitutions are all included in the scope defined by the claims of this application.

Claims

1. A saffron rhizosphere fungus, characterized in that: The rhizosphere fungus was isolated from the rhizosphere soil of saffron, a plant of the genus Crocus in the family Iridaceae, and was classified and named Cadophora malorum SRF536. It was deposited in the China General Microbiological Culture Collection Center on January 23, 2024, and the culture collection number is CGMCC No.41085.

2. An application of the saffron rhizosphere fungus according to claim 1, characterized in that: The saffron rhizosphere fungus is used to promote the growth of the model plant Arabidopsis thaliana.

3. The use according to claim 2, characterized in that: The saffron rhizosphere fungi promote the growth of Arabidopsis thaliana by promoting the growth of lateral roots, leaf growth and biomass.

4. The use according to claim 2, characterized in that: The application is specifically to inoculate saffron rhizosphere fungi into Arabidopsis culture medium for co-cultivation to promote the growth of Arabidopsis.

5. The use according to claim 4, characterized in that: The application is carried out according to the following steps: (1) selecting Arabidopsis seedlings with uniform growth and inoculating them into 1 / 2MS culture medium, with the interval between the seedlings being 0.5 to 1.0 cm; (2) The saffron rhizosphere fungi were placed 3 cm away from the Arabidopsis seedlings and cultured in a 24°C light incubator with 16 h of light and 8 h of darkness to promote the growth of Arabidopsis.

6. The use according to claim 5, characterized in that: Saffron rhizosphere fungi were inoculated in the form of bacterial cakes, and the inoculation amount of bacterial cakes was 1 bacterial cake / plate.

7. The use according to claim 5, characterized in that: The formula of 1 / 2MS culture medium is: potassium nitrate 0.95g / L, ammonium nitrate 0.825g / L, potassium dihydrogen phosphate 0.085g / L, magnesium sulfate 0.09g / L, calcium chloride 0.165g / L, potassium iodide 0.83mg / L, boric acid 6.2mg / L, manganese sulfate 16.9mg / L, zinc sulfate 8.6mg / L, sodium molybdate 0.25mg / L, copper sulfate 0.025mg / L, inositol 0.1g / L, cobalt chloride 0.025mg / L, glycine 2.0mg / L, EDTA sodium salt 37.26mg / L, vitamin B1 0.1mg / L, ferrous sulfate 15.2mg / L, niacin 0.5mg / L, vitamin B6 0.5mg / L, pH 5.80-6.00.

Images