A fungus of the genus Sarocladium ys37 and its application in promoting rice growth

Abstract

The present invention discloses a fungus of the genus Parasarocladium oryzae Verticicladium sp. YS37 and its application in promoting rice growth, belonging to the field of microbial technology. The fungus Verticicladium sp. YS37 is a new species isolated from the roots of wild rice and named Parasarocladium oryzae YS37, which is deposited in the China Center for Type Culture Collection with the deposit number of CCTCC NO: M 20242286. Parasarocladium oryzae The strain YS37 can be used to prepare bio-fertilizer. Applying the strain YS37 or its fermentation product to the roots of rice can effectively promote rice growth and increase the plant biomass. The present invention provides a new way for the development of microbial application in crop production.

Description

Technical Field

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

[0002] Rice plays a key role in global food security and the development of the agricultural economy. Relevant data shows that more than half of the world's population regards rice as the main food source. The impact of rice cultivation techniques on rice yield and quality is a key research content in the agricultural field. With the growth of the population and the limitation of resources, improving the yield and quality of rice has become the key to sustainable agricultural development.

[0003] The use of chemical fertilizers is an important means to increase rice yield, and chemical fertilizers such as nitrogen, phosphorus, and potassium are required during rice planting. However, after any type and form of chemical fertilizer is applied to farmland, it is impossible for all of it to be absorbed and utilized by plants. The unreasonable use and overuse of chemical fertilizers will cause the amount of fertilizer to exceed the retention capacity of the soil and be lost, resulting in agricultural non-point source pollution, causing eutrophication of water bodies, leading to the growth of algae, and then destroying the water environment.

[0004] Bio-bacterial fertilizer refers to a specific product containing live microorganisms and providing nutrients for plants and regulating plant growth through the specific actions of the microorganisms. Bio-bacterial fertilizer relies on the reproduction of beneficial bacteria to activate the soil, supply various nutrients required by crops, and will not damage the soil structure or pollute the environment. It is non-toxic and harmless to humans, livestock, and plants, and can protect the ecology; the fertilizer efficiency of bio-bacterial fertilizer is long-lasting, which can promote the growth and development of plants and improve the quality of crops; moreover, bio-bacterial fertilizer has a low cost and can solve the problem of pesticide residues, and it is an indispensable fertilizer for the production of green food.

[0005] An appropriate amount of bio-bacterial fertilizer can play a role in promoting seed germination, plant morphology, stress resistance, yield, and quality, etc., and can supplement and improve the nutritional status of crops, regulate plant growth, enhance plant stress resistance, and prevent and control pests and diseases. For example, the patent document CN 109536401 A discloses potassium-solubilizing bacteria Bacillus aryabhattai LZP01, phosphorus-solubilizing bacteria Bacillus pumilus LZP02, and phosphorus-dissolving bacteria Bacillus megaterium LZP03, and the combined application of the three has a significant effect on promoting the growth of rice. The patent document CN 117165449 A discloses the rice scopulariopsis Sarocladium oryzae JZ4 isolated from rice tissues has excellent biocontrol effects against rice blast.

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

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

[0008] To achieve the above purpose, the present invention adopts the following technical solutions:

[0009] The present invention isolates a new species of the genus Parasarocladium sp. from the roots of wild rice. Its biological characteristics are as follows: it grows slowly on PDA medium. The mycelia are white in the early stage, creeping on the surface of the medium, and a large number of orange spores and mucilaginous spore masses are formed on the surface of the colony in the later stage; the mycelia have vegetative branches and septa, and the conidia are oval. The ITS and LSU sequences of this strain are shown in SEQ ID NO.1 and SEQ ID NO.2 respectively. By constructing the ITS-LSU two-gene combined phylogenetic tree of this strain and its related species, this strain belongs to Parasarocladium sp., but is an independent branch (MLBP>80), and the colony morphology and hyphal spore structure are significantly different from other species of this genus. This strain is identified as a new species and named Parasarocladium oryzae sp. nov, belonging to the fungal kingdom ( Fungi ), Ascomycota ( Ascomycota ), Sordariomycetes ( Sordariomycetes ), Hypocreales ( Hypocreales ), Hypocreaceae ( Hypocreaceae ), Parasarocladium ).

[0010] Therefore, this strain is named Parasarocladium oryzae YS37, which was deposited at the China Center for Type Culture Collection on October 21, 2024. The deposit address is: Wuhan University, Wuhan, China, and the deposit number is CCTCC NO: M20242286.

[0011] Furthermore, Parasarocladium oryzae The medium for YS37 is potato dextrose agar (PDA) medium.

[0012] Furthermore, Parasarocladium oryzae The culture conditions for YS37 are dark culture at 20-25°C.

[0013] The present invention studies and finds that Parasarocladium oryzaeYS37 or its fermentation product is inoculated into the rice growth substrate, and co-cultivation can promote rice growth and increase plant biomass.

[0014] Therefore, the present invention provides the Parasarocladium oryzae application of YS37 in promoting rice growth.

[0015] Furthermore, the indexes of rice growth include plant biomass, stem and leaf length, chlorophyll content, and root length.

[0016] Furthermore, the application includes: inoculating the Parasarocladium oryzae YS37 into sterilized barley grains for cultivation to obtain a solid fermentation bacterial fertilizer, and then mixing it into the rice planting substrate to promote rice growth;

[0017] Or, inoculating the Parasarocladium oryzae YS37 into a PDB medium (liquid PDA medium without agar) for cultivation, obtaining a fermentation broth after removing the bacterial cells, and then mixing the fermentation broth into the rice culture substrate to promote rice growth.

[0018] The present invention provides Parasarocladium oryzae a preparation form of YS37, which is prepared into a solid fermentation bacterial fertilizer. The preparation method includes: inoculating the activated Parasarocladium oryzae YS37 into a PDB medium, culturing at 22 - 25 °C and 100 - 150 rpm for 3 days, adding 150 mL of mycelium suspension per 200 g of barley grains, and then culturing in the dark at 22 - 25 °C for 20 - 25 days to obtain the solid fermentation bacterial fertilizer.

[0019] The application method of the solid fermentation bacterial fertilizer can be: mixing the solid fermentation bacterial fertilizer into the seedling raising substrate, sowing the germinated rice seeds in the seedling raising substrate containing Parasarocladium oryzae YS37, and YS37 plays a role in promoting the growth of rice seedlings during the seedling raising process. Parasarocladium oryzae

[0020] The present invention also provides Parasarocladium oryzae another preparation form of YS37, which prepares its fermentation product into a nutrient solution for rice growth. The preparation method includes: inoculating the activated Parasarocladium oryzae YS37 into a PDB medium, culturing at 22 - 25 °C and 100 - 150 rpm for 15 - 20 days, and obtaining a fermentation broth after removing the bacterial cells. ​

[0021] In the present invention, the solid fermentation bacterial fertilizer or fermentation broth is applied in rice seedling raising or cultivation.

[0022] The present invention also provides a microbial inoculant for promoting the growth of rice, and the active ingredient includes the Parasarocladium oryzae YS37 or its fermentation product.

[0023] The beneficial effects of the present invention are as follows:

[0024] The present invention provides a new species YS37 of a fungus belonging to the genus Parasarocladium Sporotrichum-like sp. When the strain or its fermentation product is applied to the rice culture medium, it can effectively promote the growth of rice and increase the plant biomass. In the pot experiment, the height of the rice stems and leaves 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 fresh weight of the stems and leaves increased significantly by 41.24%, and the fresh weight of the roots increased significantly by 34.68%. In the hydroponic experiment, when the content of the YS37 fermentation broth in the culture solution was 3.3%, the height of the rice stems and leaves increased by 14.58%, the root length increased significantly by 26.62%, the fresh weight of the stems and leaves increased significantly by 35.26%, and the fresh weight of the roots increased significantly by 19.33%; when the content of the YS37 fermentation broth in the culture solution was 6.7%, the height of the rice stems and leaves increased by 22.52%, the root length increased significantly by 25.22%, the fresh weight of the stems and leaves increased significantly by 45.09%, and the fresh weight of the roots increased significantly by 38.06%. It shows that the strain YS37 and its fermentation product can promote the growth of rice, significantly increase the biomass of rice, can be used to prepare biological bacterial fertilizers, and provide a new way for the development of microorganisms applied to crop production. Description of the Drawings

[0025] Figure 1 It is a colony morphology diagram of the strain YS37 growing on the PDA medium for 20 days; among them, A is the front view of the colony, and B is the back view of the colony.

[0026] Figure 2 It is a diagram of the hypha and spore morphology of the strain YS37 under the microscope and scanning electron microscope; among them, A and B are pictures under the microscope, and C is a picture under the scanning electron microscope.

[0027] Figure 3 It is a phylogenetic tree diagram of the two-gene combination of ITS-LSU of the strain YS37. This tree is composed of ITS and LSU sequence data sets. The topological structure of the tree is the result of the BI method. The values on the branches are the BI posterior probability (BIPP) / ML bootstrap value (MLBP). The scale bar represents 0.03 base substitution sites.

[0028] Figure 4Graph showing the promotion of potted rice growth and increase in rice biomass by the solid fermentation bacterial fertilizer of strain YS37; among them, A-C are the effect graphs of strain YS37 promoting rice growth, and D-H are the statistical bar graphs of the stem and leaf height, root length, SPAD chlorophyll content, fresh weight of stem and leaf, and fresh weight of root of rice in the control group and treatment group respectively (data analyzed by independent sample t-test, * and **** respectively represent P <0.05 and P <0.0001 level of significant difference).

[0029] Figure 5 Graph showing the promotion of rice growth and increase in rice biomass by adding the 10-fold concentrated fermentation broth of strain YS37 to water for hydroponic rice; among them, A-C are the effect graphs of the concentrated fermentation broth of strain YS37 promoting rice growth, and D-G are the statistical bar graphs of the stem and leaf height, root length, fresh weight of stem and leaf, and fresh weight of root of rice in the control group and treatment group respectively (data analyzed by independent sample t-test, ** and **** respectively represent P <0.01 and P <0.0001 level of significant difference). Detailed implementation mode

[0030] 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 used to limit the scope of application of the present invention. Without departing from the spirit and essence of the present invention, any modification or replacement of the methods, steps or conditions of the present invention belongs to the scope of the present invention.

[0031] The test methods used in the following embodiments are all conventional methods unless otherwise specified; the materials, reagents, etc. used are, unless otherwise specified, reagents and materials that can be obtained from commercial channels.

[0032] Example 1: Isolation and identification of strains

[0033] 1. Isolation of strains

[0034] (1) Collect wild rice samples from Yunnan Province, and rinse the roots of wild rice with tap water to wash away the surface soil particles and attachments;

[0035] (2) Select healthy root tissues for surface disinfection, disinfect with 75% alcohol for 2 min, then disinfect with 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;

[0036] (3) Cut the disinfected roots into small segments 5 mm long;

[0037] (4)Pour the PDA medium (chloramphenicol is added to the medium to reach 50 mg / L to inhibit bacterial growth) cooled to 50 - 60 °C after sterilization into 7-cm petri dishes, 15 mL per dish; after the medium cools and solidifies, transfer the cut root segments into it with forceps for incubation;

[0038] (5)Invert the petri dishes and culture them in a constant temperature incubator at 25 °C. After culturing for 3 - 7 d, pick the hyphae of single colonies onto new PDA plates.

[0039] PDA medium: 200 g of potato, 20 g of glucose, 15 g of agar powder, add distilled water to 1000 mL, pH natural. Autoclave at 121 °C for 15 min.

[0040] A strain was isolated from the roots of common wild rice in Yunnan and named YS37.

[0041] 2. Morphological identification of the strain

[0042] The strain YS37 was inoculated in PDA medium and cultured under dark conditions at 25 ºC. The average growth rate of the hyphae was about 2.65 - 2.85 mm per day. The colony morphology and spore characteristics of the PDA medium were classified.

[0043] As Figure 1 shown, the strain YS37 grew slowly on the PDA medium. The hyphae were white in the early stage and spread on the surface of the medium. A large number of orange spores and mucilaginous spore masses were formed on the surface of the colony in the later stage.

[0044] As Figure 2 shown, under the microscope, the vegetative branches of the hyphae were septate, and the conidia were oval.

[0045] 3. Molecular identification and phylogenetic analysis

[0046] (1)DNA extraction

[0047] 1) After culturing the strain YS37 on a PDA plate at 25 ºC for 10 days, scrape the hyphae on the plate with a toothpick and put them into a 1.5-mL centrifuge tube containing 500 μL of extraction buffer and an appropriate amount of quartz sand that has been sterilized;

[0048] The formula of the extraction buffer is: 10 mM ethylenediaminetetraacetic acid, 100 mM Tris-hydrochloride, 1 M potassium chloride;

[0049] 2) Place the centrifuge tube in an MP FastPrep®-24 homogenizer and shake it at 65 Hz for 2 min to break the hyphal tissue;

[0050] 3) Centrifuge at 9000 rpm for 10 min. Use a pipette to aspirate the supernatant and transfer it to a new 1.5 mL centrifuge tube.

[0051] 4) Add isopropanol in an equal volume to the supernatant, invert the tube several times to mix thoroughly, centrifuge at 12000 rpm for 10 min, pour off the supernatant, and retain the precipitate.

[0052] 6) Add 700 μL of 70% ethanol (v / v) to dissolve impurities, mix gently, and then centrifuge at 12000 rpm for 1 min.

[0053] 7) Discard the supernatant, place the centrifuge tube upside down on absorbent paper to evaporate the 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 min. After dissolution, store the genomic DNA in a -20°C refrigerator.

[0054] (2) PCR amplification of fungal ITS and LSU rDNA genes

[0055] ITS primers: The upstream primer ITS1 sequence is: 5′-TCCGTAGGTGAACCTGCGG-3′, and the downstream primer ITS4 sequence is: 5′-TCCTCCGCTTATTGATATGC-3′;

[0056] LSU primers: The upstream primer LSU sequence is: 5′-GTACCCGCTGAACTTAAGC-3′, and the downstream primer LSU sequence is: 5′-TCCTGAGGGAAACTTCG-3′;

[0057] PCR amplification is carried out in a 30 μL reaction system, which contains: 1.5 μL of each upstream and downstream primer, 15 μL of Green TaqMix, 1.5 μL of template DNA, and 10.5 μL of ddH2O.

[0058] The PCR amplification reaction is carried out on a Longji MG96G type PCR instrument. Reaction conditions: Pre-denature at 95°C for 3 min, and then 30 cycles include: Denature at 95°C for 30 s, anneal at 55°C for 30 s, extend at 72°C for 1 min; finally, extend at 72°C for 10 min.

[0059] (3) Gene sequencing and sequence analysis

[0060] The target DNA fragment is sent to Hangzhou Youkang Biotechnology Co., Ltd. for sequencing. After the sequencing results are strictly verified, the DNA fragment sequences shown in SEQ ID NO.1 and SEQ ID NO.2 are obtained, which are the ITS and LSU gene sequences of strain YS37.

[0061] On the NCBI website, the nucleotide sequences of ITS and LSU of the measured YS37 strain were searched and aligned with homologous or similar nucleotide sequences in the GenBank database using BLAST. BLAST alignment found that the ITS sequence had 100% similarity with the strain Parasarocladium sp. YF-56 with the accession number OQ832464, and 96.64% similarity with the strain Parasarocladium tasmanniae CBS 146807 with the accession number NR_171987. Through sequence comparison, the YS37 strain belongs to Parasarocladium sp.

[0062] Phylogenetic analysis

[0063] On NCBI, the ITS and LSU sequences of the related genera of strain YS37 were searched, and a phylogenetic tree of the ITS-LSU two-gene combination of strain YS37 and its related genera was constructed. The results showed that there were a total of 1543 bases at the combined information sites of the two genes, including 477 parsimony-informative sites, 683 variable information sites, and 875 conserved sites. Through jModel Test2.1.7, GTR+I+G and GTR+G were calculated as the best models for Bayesian (BI) and maximum likelihood method (ML) respectively, and phylogenetic trees of the Bayesian method (BI) and maximum likelihood method (ML) were established. The topological structures of the phylogenetic trees constructed by the BI and ML methods were highly similar. Therefore, only the BI tree is shown Figure 3 herein. From the phylogenetic tree, strain YS37 belongs to Parasarocladium sp., but it is not in the same branch as P. tasmanniae and there are also significant differences in the colony morphology and hyphal spore morphology between strain YS37 and P. tasmanniae .

[0064] Based on comprehensive phylogenetic analysis, morphological and biological characteristics, strain YS37 was defined as a new species Parasarocladium oryzae sp. nov, belonging to the fungal kingdom ( Fungi ), Ascomycota ( Ascomycota ), Sordariomycetes ( Sordariomycetes ), Hypocreales ( Hypocreales ), Hypocreaceae ( Hypocreaceae ), Parasarocladium ).

[0065] Therefore, this strain was named Parasarocladium oryzae YS37, which was deposited in the China Center for Type Culture Collection on October 21, 2024. The deposit address is: Wuhan University, Wuhan, China, and the deposit number is CCTCC NO: M20242286. It was detected as viable on October 28, 2024.

[0066] Example 2: Promoting effect of solid-fermented bacterial fertilizer of YS37 strain on the growth of potted rice

[0067] 1. Rice seed germination: Wash rice seeds successively with clean water, 1% NaClO (soak for 10 min), and sterile water. Put the washed seeds into a glass basin containing wet blotting paper and germinate them in the dark at 37°C for 2 days.

[0068] 2. Preparation of bacterial fertilizer: Culture the YS37 strain on PDA medium for 7 days. Punch out bacterial cakes and place them in 150 mL of PDB medium, and culture at 25°C and 150 rpm for 3 days. Then pour the mycelial suspension into autoclaved (121°C for 30 min) barley grains (add 150 mL of bacterial liquid to every 200 g of barley grains) and ferment at 25°C for 20 days.

[0069] 3. Potted planting: Let the fermented bacterial fertilizer dry naturally. Mix the dried bacterial fertilizer with the rice planting substrate and put it into planting pots. Plant the germinated rice seeds into the pots containing the fermented bacterial fertilizer (30 seeds and 40 g of fertilizer per pot). Use sterile barley seed fertilizer as the control group.

[0070] 4. Phenotypic analysis of rice plants: After culturing the rice for 24 days, measure the chlorophyll content of rice leaves with a chlorophyll content meter SPAD-502 (Japan). Pull out the rice seedlings from the soil substrate together with the roots, wash them repeatedly with clean water, and dry the water on the rice plants with blotting paper. Measure the height of the rice stems and leaves, root length, fresh weight of the stems and leaves, and roots respectively.

[0071] 5. Result analysis: As Figure 4 shown, in the potted experiment, the height of the rice stems and leaves 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 fresh weight of the stems and leaves increased significantly by 41.24%, and the fresh weight of the roots increased significantly by 34.68%.

[0072] Example 3: Promoting effect of the fermentation broth of YS37 strain on the growth of hydroponic rice

[0073] 1. Inoculate the YS37 strain into 400 mL of PDB medium / 500 mL conical flask, culture at 25°C and 150 rpm for 15 d, centrifuge to obtain the supernatant as the fermentation broth. Use an EYELA rotary evaporator to rotary evaporate the fermentation broth at 65°C to remove the excess water and concentrate the fermentation broth by 10 times. Take 0.5 mL and 1 mL of the concentrated fermentation broth respectively and add them to the culture flask, and make up the volume to 150 mL with water to prepare culture solutions with fermentation broth contents of 3.3% and 6.7% respectively. Use the culture solution without adding the fermentation broth as the control.

[0074] 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 the culture bottle (20 seeds per bottle), and after culturing for 15 days (replenish the culture solution once during the culturing process), take pictures and record, and measure the stem and leaf height, root length, fresh weight of stem and leaf, and fresh weight of root of the rice seedlings.

[0075] 3. Result analysis: As Figure 5 shown, in the hydroponic experiment, when the content of YS37 fermentation broth in the culture solution was 3.3%, the stem and leaf height of the rice increased by 14.58%, the root length increased significantly by 26.62%, the fresh weight of stem and leaf increased significantly by 35.26%, and the fresh weight of root increased significantly by 19.33%. When the content of YS37 fermentation broth in the culture solution was 6.7%, the stem and leaf height of the rice increased by 22.52%, the root length increased significantly by 25.22%, the fresh weight of stem and leaf increased significantly by 45.09%, and the fresh weight of root increased significantly by 38.06%.

[0076] Although the embodiments of the present invention have been described, for those of ordinary skill in the art, it can be understood that some changes and modifications can be made to these 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 Parasarocladium sp. YS37, characterized in that This bacterium was isolated from the roots of wild rice and named Parasarocladium oryzae YS37, and is deposited in the China Center for Type Culture Collection with the deposit number CCTCC NO: M 20242286.

2. The fungus Parasarocladium sp. YS37 of the genus The Parasarocladium oryzae culture medium of YS37 is potato dextrose agar medium.

3. The Cladosporium sp. YS37 as claimed in claim 1 or 2, characterized in that, Parasarocladium sp.), The described Parasarocladium oryzae The culture conditions of YS37 are dark culture at 20 - 25°C.

4. Use of the fungus Parasarocladium sp. YS37 of the genus Verticicladium in promoting the growth of rice as described in any one of claims 1-3.

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

6. The application according to claim 4, characterized in that The application includes: inoculating the Parasarocladium oryzae YS37 into sterilized barley grains, culturing to obtain a solid fermentation bacterial fertilizer, and then mixing it into a rice planting substrate to promote rice growth; Alternatively, inoculate the Parasarocladium oryzae YS37 into PDB medium for culture, obtain the fermentation broth after removing the bacteria, and then mix the fermentation broth into the rice culture medium to promote the growth of rice.

7. The application according to claim 6, wherein The solid fermentation bacterial fertilizer or fermentation broth is applied in rice seedling raising or cultivation.

8. The application according to claim 6, wherein The preparation method of the solid fermentation bacterial fertilizer includes: inoculating the activated Parasarocladium oryzae YS37 into the PDB medium, culturing at 22-25 °C and 100-150 rpm for 3 days, adding 150 mL of mycelium suspension per 200 g of barley grains, and then culturing in the dark at 22-25 °C for 20-25 days to obtain the solid fermentation bacterial fertilizer.

9. The application according to claim 6, characterized in that, The preparation method of the fermentation broth includes: inoculating the activated Parasarocladium oryzae YS37 into a PDB medium, culturing at 22-25 °C and 100-150 rpm for 15-20 days, and obtaining the fermentation broth after removing the bacterial cells.

10. A microbial inoculant for promoting rice growth, characterized in that, The active ingredient includes those described in any one of claims 1-3 Parasarocladium oryzae YS37 or its fermentation broth.

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