Cladosporium sp. Fungus YS37 and application thereof in promoting rice growth

Abstract

The invention discloses a cladosporium sp. Fungus YS37 and application thereof in promoting rice growth, and belongs to the technical field of microorganisms. The cladosporioides sp. Fungus YS37 is a new species separated from the root system of wild rice, is named as Parasarocladium oryzae YS37, is preserved in the China Center for Type Culture Collection (CCTCC), and has a preservation number of CCTCC NO: M 20242286, and the cladosporioides sp. Fungus YS37 is a new species which is separated from the root system of wild rice and is named as Parasarocladium oryzae YS37. The Parasarocladium oryzae YS37 strain can be used for preparing a biological bacterial fertilizer, and when the Parasarocladium oryzae YS37 strain or a fermentation product of the Parasarocladium oryzae YS37 strain is applied to a rice root system, the growth of rice can be effectively promoted, and the plant biomass is increased. The invention provides a new way for application of microorganisms to development of crop production.

Description

Technical Field

[0001] The present invention relates to the technical field of microorganisms, and in particular to a fungus of the genus Cladosporium ( Parasarocladium sp.) YS37 and its application in promoting rice growth. Background Art

[0002] Rice plays a key role in global food security and agricultural economic development. Relevant data show that more than half of the world's population relies on rice as their main food source. The impact of rice cultivation technology on rice yield and quality is a key research topic in the agricultural field. With the growth of population and limited resources, improving rice yield and quality has become the key to sustainable agricultural development.

[0003] The use of chemical fertilizers is an important means to increase rice yields. Chemical fertilizers such as nitrogen, phosphorus and potassium are needed in the rice planting process. However, it is impossible for all types and forms of chemical fertilizers to be absorbed and utilized by plants after being applied to farmland. Irrational use and excessive use of chemical fertilizers will cause the amount of fertilizer to exceed the soil's retention capacity and be lost, forming agricultural non-point source pollution, causing eutrophication of water bodies, leading to algae growth, and then damaging the water environment.

[0004] Biofertilizer refers to a specific product that contains living microorganisms and provides nutrition to plants and regulates plant growth through the specific effects of microorganisms. Biofertilizer relies on the proliferation of beneficial bacteria to activate the soil and provide various nutrients needed by crops. It does not destroy the soil structure or pollute the environment. It is non-toxic and harmless to humans, livestock and plants and can protect the ecology. Biofertilizer has a long-lasting effect and can promote plant growth and development and improve crop quality. In addition, biofertilizer is low-cost and can solve the problem of pesticide residues. It is an indispensable fertilizer for the production of green food.

[0005] The appropriate amount of biological fertilizer can improve seed germination, plant morphology, stress resistance and yield quality, and can supplement and improve crop nutrition, regulate plant growth, enhance plant stress resistance and prevent diseases and insect pests. For example, patent document CN 109536401 A discloses the screening of potassium-dissolving bacteria from black soil in rice root system. Bacillus aryabhattai LZP01, phosphate-solubilizing bacteria Bacillus pumilus LZP02, phosphate-dissolving bacteria Bacillus megaterium LZP03, the combination of the three has a significant effect on promoting rice growth. Patent document CN 117165449 A discloses a fungus isolated from rice tissue Sarocladium oryzae JZ4 has excellent biocontrol effect against rice blast.

[0006] As an important aspect of sustainable agriculture, the microbial fertilizer industry will exert its great potential for application in agriculture. Therefore, screening beneficial microorganisms and developing biofertilizers is an important method to promote safe and efficient production of crops. Summary of the invention

[0007] The purpose of the present invention is to provide a microorganism that can promote the growth of rice, and to develop it into a biological fertilizer for safe and efficient production of rice.

[0008] To achieve the above object, the present invention adopts the following technical solution: The present invention isolates a fungus of the genus Cladosporium from the root system of wild rice ( Parasarocladium sp.) with the following biological characteristics: it grows slowly on PDA medium, with white hyphae in the early stage, crawling on the surface of the medium, and forming a large number of orange spores and myxospores on the surface of the colony in the later stage; the hyphae have vegetative branches and septa, and the conidia are oval. The ITS and LSU sequences of the strain are shown in SEQ ID NO.1 and SEQ ID NO.2, respectively. By constructing the ITS-LSU two-gene joint phylogenetic tree of the strain and its closely related species, the strain belongs to Parasarocladium sp., but it was an independent branch (MLBP>80), and its colony morphology and hyphae and spore structure were significantly different from those of other species of the genus. This strain was identified as a new species and named Parasarocladium oryzae sp. nov, belonging to the kingdom Fungi ( Fungi )、Ascomycota( Ascomycota )、Faecalis( Sordariomycetes )、Hypocreales( Hypocreales )、Hypocreaceae( Hypocreaceae ), Cladosporium spp. Parasarocladium ).

[0009] Therefore, the strain was named Parasarocladium oryzae YS37, deposited in China Center for Type Culture Collection on October 21, 2024, deposit address: Wuhan University, Wuhan, China, with the deposit number CCTCC NO: M20242286.

[0010] Furthermore, Parasarocladium oryzae The culture medium of YS37 is potato dextrose agar (PDA) medium.

[0011] Furthermore, Parasarocladium oryzae The culture condition of YS37 is 20-25°C in the dark.

[0012] The present invention has found that Parasarocladium oryzaeYS37 or its fermentation product is inoculated into a rice growth matrix, and can promote rice growth and increase plant biomass through co-cultivation.

[0013] Therefore, the present invention provides the Parasarocladium oryzae Application of YS37 in promoting rice growth.

[0014] Furthermore, the rice growth indicators include plant biomass, stem and leaf length, chlorophyll content, and root length.

[0015] Further, the application includes: Parasarocladium oryzae YS37 was inoculated into sterilized barley grains to obtain solid fermentation fertilizer, which was then mixed into rice planting substrate to promote rice growth; Alternatively, Parasarocladium oryzae YS37 was inoculated into PDB medium (liquid PDA medium without agar) and cultured. The bacterial cells were removed to obtain the fermentation liquid, which was then mixed into the rice culture matrix to promote rice growth.

[0016] The present invention provides Parasarocladium oryzae A preparation form of YS37 is prepared into a solid fermentation fertilizer. The preparation method includes: Parasarocladium oryzae YS37 was inoculated into PDB medium, cultured at 22-25°C and 100-150 rpm for 3 days, 150 mL of mycelium suspension was added for every 200 g of barley grains, and then cultured in the dark at 22-25°C for 20-25 days to obtain solid fermentation fertilizer.

[0017] The solid fermented bacterial fertilizer can be applied in the following manner: the solid fermented bacterial fertilizer is mixed into a seedling raising substrate, and the germinated rice seeds are sown in a medium containing Parasarocladium oryzae In the seedling medium of YS37, during the seedling raising process Parasarocladium oryzae YS37 plays a role in promoting the growth of rice seedlings.

[0018] The present invention also provides Parasarocladium oryzae Another preparation form of YS37 is to prepare the fermentation product thereof into a nutrient solution for rice growth. The preparation method comprises: Parasarocladium oryzae YS37 was inoculated into PDB medium, cultured at 22-25°C and 100-150 rpm for 15-20 days, and the bacterial cells were removed to obtain the fermentation liquid.

[0019] In the present invention, the solid fermented bacterial fertilizer or fermented liquid is applied during rice seedling raising or cultivation.

[0020] The present invention also provides a microbial agent for promoting rice growth, the active ingredients of which include the Parasarocladium oryzae YS37 or a fermentation product thereof.

[0021] The present invention has the following beneficial effects: The present invention provides a strain of Cladosporium sp. Parasarocladium sp.) new species YS37, the strain or its fermentation products applied to the rice culture medium can effectively promote rice growth and increase plant biomass. In the pot experiment, the stem and leaf height of rice co-cultured with YS37 increased significantly by 25.77%, the root length increased significantly by 16.76%, the chlorophyll content increased significantly by 14.56%, the stem and leaf fresh weight increased significantly by 41.24%, and the root fresh weight increased significantly by 34.68%. In the hydroponic experiment, when the content of YS37 fermentation liquid in the culture medium was 3.3%, the stem and leaf height of rice increased by 14.58%, the root length increased significantly by 26.62%, the stem and leaf fresh weight increased significantly by 35.26%, and the root fresh weight increased significantly by 19.33%; when the content of YS37 fermentation liquid in the culture medium was 6.7%, the stem and leaf height of rice increased by 22.52%, the root length increased significantly by 25.22%, the stem and leaf fresh weight increased significantly by 45.09%, and the root fresh weight increased significantly by 38.06%. The results showed that strain YS37 and its fermentation products can promote rice growth, significantly increase rice biomass, and can be used to prepare biofertilizer, providing a new way for the development of microorganisms in crop production. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Figure 1 The colony morphology of strain YS37 grown on PDA medium for 20 days; A is the front view of the colony, and B is the back view of the colony.

[0023] Figure 2 The hyphae and spore morphologies of strain YS37 under a microscope and a scanning electron microscope; A and B are images under a microscope, and C is an image under a scanning electron microscope.

[0024] Figure 3 This is the ITS-LSU two-gene joint phylogenetic tree of strain YS37. The tree is composed of ITS and LSU sequence data sets. The topology of the tree is the result of the BI method. The values ​​on the branches are BI posterior probability (BIPP) / ML bootstrap value (MLBP). The scale represents 0.03 base substitution sites.

[0025] Figure 4The results of solid fermentation fertilizer of strain YS37 promoting the growth of potted rice and increasing rice biomass are shown in the figure; AC is the effect diagram of strain YS37 promoting rice growth, and DH is the statistical bar graph of stem and leaf height, root length, SPAD chlorophyll content, stem and leaf fresh weight and root fresh weight of rice in the control group and treatment group respectively (independent sample t-test analysis data, * and **** represent P <0.05 and P <0.0001 level).

[0026] Figure 5 The results of adding the fermentation liquid of strain YS37 concentrated 10 times to hydroponic rice in water to promote rice growth and increase rice biomass are shown in the figure; AC is the effect of the fermentation liquid of strain YS37 concentrated liquid on promoting rice growth, and DG is the statistical bar graph of the stem and leaf height, root length, stem and leaf fresh weight and root fresh weight of rice in the control group and the treatment group, respectively (independent sample t-test analysis data, ** and **** represent P <0.01 and P <0.0001 level). DETAILED DESCRIPTION

[0027] The present invention will be further described below in conjunction with specific embodiments. The following embodiments are only used to illustrate the present invention and are not intended to limit the scope of application of the present invention. Without departing from the spirit and essence of the present invention, modifications or replacements made to the inventive method, steps or conditions all belong to the scope of the present invention.

[0028] Unless otherwise specified, the experimental methods used in the following examples are all conventional methods; the materials and reagents used are reagents and materials that can be obtained from commercial channels unless otherwise specified.

[0029] Example 1: Isolation and identification of bacterial species 1. Isolation of bacterial strains (1) Wild rice samples were collected from Yunnan Province, and the roots of the wild rice were washed with tap water to remove the soil particles and attachments on the surface; (2) Select healthy root tissue for surface disinfection. Use 75% alcohol for 2 min, then 1% sodium hypochlorite for 4-5 min, and finally wash with sterile distilled water 4-5 times to wash away the residual sodium hypochlorite solution. (3) Cut the disinfected roots into small segments of 5 mm in length; (4) Pour the sterilized PDA culture medium (add chloramphenicol to the culture medium to reach 50 mg / L to inhibit bacterial growth) cooled to 50-60°C into 7 cm culture dishes, 15 mL per dish; after the culture medium cools and solidifies, use tweezers to transfer the cut root segments into the culture medium for incubation; (5) Place the culture dish upside down in a constant temperature incubator at 25°C. After culturing for 3-7 days, pick out the hyphae of a single colony and place it on a new PDA plate.

[0030] PDA medium: 200 g potato, 20 g glucose, 15 g agar powder, add distilled water to 1000 mL, pH natural. Autoclave at 121℃ for 15 min.

[0031] A strain was isolated from the roots of common wild rice in Yunnan and named YS37.

[0032] 2. Identification of bacterial species morphology Strain YS37 was inoculated in PDA medium and cultured in the dark at 25ºC. The average mycelial growth rate was about 2.65-2.85 mm per day. The colony morphology and spore characteristics of PDA medium were classified.

[0033] like Figure 1 As shown, strain YS37 grows slowly on PDA medium. In the early stage, the hyphae are white and crawl on the surface of the medium. In the later stage, a large number of orange spores and myxospores form on the surface of the colony.

[0034] like Figure 2 As shown, under the microscope, the hyphae are branched and septate, and the conidia are oval.

[0035] 3. Molecular identification and phylogenetic analysis (1) DNA extraction 1) After culturing the YS37 strain on a PDA plate at 25°C for 10 days, scrape the mycelium from the plate with a toothpick and place it in a sterilized 1.5 mL centrifuge tube containing 500 μL extraction buffer and an appropriate amount of quartz sand; The formula of the extraction buffer was: 10 mM EDTA, 100 mM Tris-HCl, 1 M KCl; 2) Place the centrifuge tube in an MP FastPrep®-24 homogenizer and shake at 65 Hz for 2 min to break up the mycelial tissue; 3) Centrifuge at 9000 rpm for 10 min, pipette the supernatant and add it to a new 1.5 mL centrifuge tube; 4) Add an equal volume of isopropanol to the supernatant, mix by inverting several times, centrifuge at 12,000 rpm for 10 min, discard the supernatant, and retain the precipitate; 6) Add 700 μL 70% ethanol (v / v) to dissolve impurities, mix gently, and centrifuge at 12,000 rpm for 1 min; 7) Discard the supernatant and place the centrifuge tube upside down on absorbent paper to evaporate excess ethanol. The white precipitate is the genomic DNA to be extracted. Add 50 μL of sterile water and dissolve it at room temperature for 15 minutes. After the dissolution is completed, store the genome in a -20°C refrigerator.

[0036] (2) PCR amplification of fungal ITS and LSU rDNA genes ITS primers: the sequence of the upstream primer ITS1 is: 5′-TCCGTAGGTGAACCTGCGG-3′, the sequence of the downstream primer ITS4 is: 5′-TCCTCCGCTTATTGATATGC-3′; LSU primers: The sequence of the upstream primer LSU is: 5′-GTACCCGCTGAACTTAAGC-3′, and the sequence of the downstream primer LSU is: 5′-TCCTGAGGGAAACTTCG-3′; PCR amplification was performed in a 30 μL reaction system, which contained: 1.5 μL of upstream and downstream primers, 15 μL of Green TaqMix, 1.5 μL of template DNA, and ddH 2 O 10.5 μL.

[0037] PCR amplification was performed on a Longji MG96G PCR instrument. The reaction conditions were: 95°C pre-denaturation for 3 min, followed by 30 cycles of: 95°C denaturation for 30 s, 55°C annealing for 30 s, 72°C extension for 1 min, and finally 72°C post-extension for 10 min.

[0038] (3) Gene sequencing and sequence analysis The target DNA fragment was sent to Hangzhou Youkang Biotechnology Co., Ltd. for sequencing. After strict verification of the sequencing results, the DNA fragment sequences shown as SEQ ID NO.1 and SEQ ID NO.2 were obtained, namely the ITS and LSU gene sequences of strain YS37.

[0039] On the NCBI website, the ITS and LSU nucleotide sequences of the YS37 strain were searched and compared with homologous or similar nucleotide sequences in the GenBank database using BLAST. BLAST comparison revealed that the ITS sequence was similar to the strain with accession number OQ832464. Parasarocladium sp. YF-56 has a 100% similarity to the strain with accession number NR_171987 Parasarocladium tasmanniae CBS 146807 has a similarity of 96.64%. Through sequence comparison, strain YS37 belongs to Parasarocladium sp.

[0040] Phylogenetic analysis The ITS and LSU sequences of the closely related genera of strain YS37 were searched on NCBI, and a phylogenetic tree of the ITS-LSU gene combination of strain YS37 and its closely related genera was constructed. The results showed that there were 1543 bases in total for the combined information sites of the two genes, including 477 parsimonious information sites, 683 variable information sites, and 875 conserved sites. Using jModel Test2.1.7, it was calculated that GTR+I+G and GTR+G were the best models for Bayesian (BI) and maximum likelihood (ML), respectively, and phylogenetic trees of Bayesian (BI) and maximum likelihood (ML) methods were established. The topological structures of the phylogenetic trees constructed by the BI and ML methods are highly similar. Therefore, in Figure 3 Only the BI tree is shown in the figure. From the phylogenetic tree, strain YS37 belongs to Parasarocladium sp., but it is related to P. tasmanniae Not in the same branch and strains YS37 and P. tasmanniae The colony morphology and mycelial spore morphology are also very different.

[0041] Comprehensive phylogenetic analysis, morphological and biological characteristics defined strain YS37 as a new species Parasarocladium oryzae sp. nov, belongs to the kingdom Fungi ( Fungi )、Ascomycota( Ascomycota )、Faecalis( Sordariomycetes )、Hypocreales( Hypocreales )、Hypocreaceae( Hypocreaceae ), Cladosporium spp. Parasarocladium ).

[0042] Therefore, the strain was named Parasarocladium oryzae YS37, deposited in China Center for Type Culture Collection on October 21, 2024, deposit address: Wuhan University, Wuhan, China, deposit number: CCTCC NO: M20242286, detected as alive on October 28, 2024.

[0043] Example 2: Effect of solid fermentation fertilizer of YS37 strain on the growth of potted rice 1. Rice seed germination: Wash the rice seeds with clean water, 1% NaClO (soak for 10 min), and sterile water in turn. Place the washed seeds in a glass basin containing moistened absorbent paper and germinate at 37°C in the dark for 2 days.

[0044] 2. Preparation of bacterial fertilizer: YS37 strain was cultured in PDA medium for 7 days, and bacterial cake was placed in 150 mL PDB medium, and cultured at 25℃ 150 rpm for 3 days. Then the mycelium suspension was poured into barley grains after high-pressure sterilization (121℃ 30 min) (150 mL bacterial solution was added for every 200 g barley grains), and fermented at 25℃ for 20 days.

[0045] 3. Potted Planting: Let the fermented bacterial fertilizer dry naturally, mix the dried bacterial fertilizer with the rice planting substrate and put it into the planting pot, plant the germinated rice seeds in the pot containing the fermented bacterial fertilizer (30 seeds and 40 g fertilizer per pot). Use sterile barley seed fertilizer as the control group.

[0046] 4. Analysis of rice plant phenotype: After culturing the rice for 24 days, the chlorophyll content of the rice leaves was measured using the chlorophyll content meter SPAD-502 (Japan). The rice seedlings were pulled out of the soil matrix with their roots, washed repeatedly with clean water, and the moisture on the rice plants was absorbed with absorbent paper. The height of the rice stems and leaves, root length, and fresh weight of the stems and leaves and roots were measured respectively.

[0047] 5. Result analysis: Figure 4 As shown, in the pot experiment, the stem and leaf height of rice co-cultured with YS37 increased significantly by 25.77%, the root length increased significantly by 16.76%, the chlorophyll content increased significantly by 14.56%, the stem and leaf fresh weight increased significantly by 41.24%, and the root fresh weight increased significantly by 34.68%.

[0048] Example 3: Effect of YS37 fermentation broth on the growth of hydroponic rice 1. YS37 strain was inoculated into 400 mL PDB medium / 500 mL conical flask, cultured at 25℃ 150 rpm for 15 days, and the supernatant was centrifuged as the fermentation broth. The fermentation broth was evaporated by EYELA rotary evaporator at 65℃ to remove excess water, and the fermentation broth was concentrated 10 times. 0.5 mL and 1 mL of the concentrated fermentation broth were added to the culture flask respectively, and water was added to make the volume 150 mL to prepare the culture broth with a fermentation broth content of 3.3% and 6.7% respectively. The culture broth without fermentation broth was used as the control.

[0049] 2. Sprinkle a small amount of perlite on the surface of the prepared culture solution (to prevent the rice seeds from sinking to the bottom), transfer the germinated rice seeds into culture bottles (20 seeds per bottle), and culture for 15 days (replenish the culture solution once during the culture process). Then take photos and measure the stem and leaf height, root length, stem and leaf fresh weight, and root fresh weight of the rice seedlings.

[0050] 3. Result analysis: Figure 5As shown in the figure, in the hydroponic experiment, when the content of YS37 fermentation liquid in the culture medium was 3.3%, the stem and leaf height of rice increased by 14.58%, the root length increased significantly by 26.62%, the stem and leaf fresh weight increased significantly by 35.26%, and the root fresh weight increased significantly by 19.33%. When the content of YS37 fermentation liquid in the culture medium was 6.7%, the stem and leaf height of rice increased by 22.52%, the root length increased significantly by 25.22%, the stem and leaf fresh weight increased significantly by 45.09%, and the root fresh weight increased significantly by 38.06%.

[0051] Although the embodiments of the present invention have been described, it will be appreciated by those skilled in the art that some changes and modifications may be made to the embodiments without departing from the principles and spirit of the present invention. The scope of the present invention is defined by the appended claims and their equivalents.

Claims

1. A fungus of the genus Cladosporium ( Parasarocladium sp.) YS37, characterized in that The fungus was isolated from wild rice roots and named Parasarocladium oryzae YS37 is deposited in China Center for Type Culture Collection with the deposit number CCTCC NO: M 20242286.

2. The Cladosporium fungus according to claim 1 ( Parasarocladium sp.) YS37, characterized in that Said Parasarocladium oryzae The culture medium of YS37 is potato dextrose agar.

3. The Cladosporium fungus according to claim 1 or 2 ( Parasarocladium sp.) YS37, characterized in that Said Parasarocladium oryzae The culture condition of YS37 is 20-25°C in the dark.

4. The Cladosporium fungus according to any one of claims 1 to 3 ( Parasarocladium sp.) YS37 in promoting rice growth.

5. The use according to claim 4, characterized in that The rice growth indicators include plant biomass, stem and leaf length, chlorophyll content, and root length.

6. The use according to claim 4, characterized in that The application includes: Parasarocladium oryzae YS37 was inoculated into sterilized barley grains to obtain solid fermentation fertilizer, which was then mixed into rice planting substrate to promote rice growth; Alternatively, Parasarocladium oryzae YS37 was inoculated into PDB medium for cultivation, and the fermentation liquid was obtained after removing the bacterial cells, and then the fermentation liquid was mixed into the rice culture matrix to promote the growth of rice.

7. The use according to claim 6, characterized in that The solid fermented bacterial fertilizer or fermented liquid is applied during rice seedling raising or cultivation.

8. The use according to claim 6, characterized in that The preparation method of the solid fermentation fertilizer comprises: Parasarocladium oryzae YS37 was inoculated into PDB medium, cultured at 22-25°C and 100-150 rpm for 3 days, 150 mL of mycelium suspension was added for every 200 g of barley grains, and then cultured in the dark at 22-25°C for 20-25 days to obtain solid fermentation fertilizer.

9. The use according to claim 6, characterized in that The method for preparing the fermentation broth comprises: Parasarocladium oryzae YS37 was inoculated into PDB medium, cultured at 22-25°C and 100-150 rpm for 15-20 days, and the bacterial cells were removed to obtain the fermentation liquid.

10. A microbial agent for promoting rice growth, characterized in that: The active ingredient comprises the Parasarocladium oryzae YS37 or a fermentation product thereof.

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