A polysporic root cyst fungus CAUBW20 and its application in resisting root-knot nematodes, promoting plant growth and nutrient absorption

Abstract

The present invention discloses a multisporic root cyst fungus (Rhizoglomus multiployspora) CAUBW 20 and its application in resisting root-knot nematodes, promoting plant growth and nutrient absorption, and relates to the strain and its application technology field. The multisporic root cyst fungus (Rhizoglomus multiployspora) strain was deposited in the General Microbiological Center of the China Culture Collection Committee on May 8, 2023, with a deposit number of CGMC C NO: 40588, and a deposit address of No. 3, Yard No. 1, Beichen West Road, Chaoyang District, Beijing. It is classified and named as: multisporic root cyst fungus Rhizoglomus multiployspora, and the strain name is CAUBW20; the present invention also provides a method for preparing a bacterial agent of the strain and an application thereof. The bacterial agent containing the strain provided by the present invention significantly improves the growth, nutrient absorption and root-knot nematode resistance of vegetable crops such as tomatoes, and has important application value and application prospects in the resistance of vegetable crops to root knot nematodes.

Description

Technical Field

[0001] The invention relates to the technical field of screening and application of Rhizoglomus multiployspora, and more particularly to a Rhizoglomus multiployspora CAUBW20 and its application in resisting root-knot nematodes and promoting plant growth and nutrient absorption. Background Art

[0002] Root-knot nematodes can infect more than 2,000 plant species and are one of the pathogens with the greatest impact on the agricultural economy. Once they infect the root system, they can seriously harm the plants and cause a significant reduction in yield. Currently, vegetable production uses large-scale facility planting and intensive cultivation. While achieving high yields, the population density of root-knot nematodes in the soil increases year by year due to the cut-off of winter soil freezing and the reduction of crop rotations, posing a continuous threat to vegetable production.

[0003] Root-knot nematodes are spread by larvae and eggs attached to seeds, farming tools, seedling transplanting, and tuber transportation. Once infected, it is difficult to eliminate. The most effective control method at present is to use pesticides to treat the soil. However, the treatment process is complicated, costly, and easy to develop drug resistance. In addition, these chemicals are toxic, causing environmental pollution and food safety risks. Large-scale and repeated use leads to an imbalance in the composition of soil biological communities, and nematodes develop drug resistance. Therefore, sustainable green alternative technologies are urgently needed in production, and microorganisms are one of the key technical solutions to this problem.

[0004] Therefore, the Rhizoglomus multiployspora strain CAUBW20 provided by the present invention and its application in crop planting are important technical solutions to solve this problem urgently needed in the art. Summary of the invention

[0005] In view of this, the technical solution of the present invention is as follows:

[0006] An arbuscular mycorrhizal fungus, which belongs to the genus Rhizoglomus, species name: Rhizoglomus multiployspora, strain name: CAUBW20, deposited in the General Microbiological Center of the China Culture Collection Administration on May 8, 2023, with a deposit number of CGMCC NO.40588, and a deposit address of No. 3, Yard No. 1, Beichen West Road, Chaoyang District, Beijing. It is classified and named Rhizoglomus multiployspora. According to the latest international biological classification standards in 2024, it belongs to the genus Rhizophagus of the phylum Glomeromycotina, class Glomeromycetes, order Glomerales, family Glomeromyceae. As an inventive concept identical to the above-mentioned technical scheme, the present invention also requests protection for the use of the multisporous root cyst fungus (Rhizoglomus multiployspora) CAUBW20 in the preparation of an antagonistic root-knot nematode bacterial agent;

[0007] The present invention also claims to protect the use of the multisporous root cyst fungus (Rhizoglomus multiployspora) CAUBW20 in the preparation of a microbial agent for increasing vegetable biomass, yield and nutrient absorption;

[0008] The present invention also claims a bacterial agent that antagonizes root-knot nematodes and improves nutrient absorption, yield and biomass of vegetables, wherein the bacterial agent comprises Rhizoglomus multiployspora CA UBW20;

[0009] The present invention also requests protection for the preparation method of the above-mentioned bacterial agent, and its production process is: using plants such as tomatoes and carrots as host propagation materials, using nutrient soil and sieved river sand as culture matrix, sterilizing at a high temperature of 85° for 48 hours; then adding 10-50g of the pre-expansion bacterial agent to 1L of the above-mentioned sterilized matrix, mixing, and placing on a plate; the host sowing amount is 10-20 sterilized seeds / pot, the seeds are sterilized by soaking in 5-8% sodium hypochlorite solution for 10 minutes, and the bacterial sand is harvested after culturing for 2-4 months.

[0010] It can be seen from the above technical scheme that the technical effect achieved by the present invention is: the present invention screens and discovers a multisporic root cyst fungus (Rhizoglomus multiployspora), prepares it into a microbial agent, and the provided microbial agent significantly promotes the growth, nutrient absorption and root nematode resistance of vegetable crops such as tomatoes, and has important application value and application prospects in the resistance of vegetable crops to root knot nematodes. BRIEF DESCRIPTION OF THE DRAWINGS

[0011] In order to more clearly illustrate the embodiments of the present invention or the prior art solutions, the drawings described below are only embodiments of the present invention. For ordinary technicians in this field, other technical drawings can be obtained based on the drawings without paying any creative work.

[0012] Figure 1 The morphological characteristics of Rhizoglomus multiployspora CAUBW20 of the present invention are shown in Figures A, B, C, and D. The spores are all floated in a floating carrier (PLVG) solution of "polyvinyl alcohol, lactic acid, and glycerol". A and B are the morphology of Rhizoglomus multiployspora CAUBW20 in the root, and C and D are the morphology of the spores when they are crushed in the floating carrier. The spores in E and F are the crushed morphology of the spores when they are floated in a mixed solution of PLVG solution and Melzer reagent (Melzer reagent) of "water, chloral, iodine, and potassium iodide" (v:v=1:1). A and B are photos of the spores magnified 20 times, and C, D, E, and F are photos of the spores magnified 100 times.

[0013] Figure 2 It is the Bayesian (MrByes) maximum likelihood phylogenetic tree diagram of the rDNA SSU-ITS-LSU (18S-ITS-28S) sequence of Rhizoglomus multiployspora CAUBW20;

[0014] Figure 3 The Bayesian MrByes maximum likelihood phylogenetic tree diagram of the rDNA RPB1 sequence of Rhizoglomus multiployspora CAUBW20;

[0015] Figure 4 The growth of tomato plants and the plants infected by root-knot nematodes; CK is the blank control, RK (single plant inoculated with 500 2nd instar larvae of root-knot nematodes), PL (Paecilomyces lilacinus + inoculated with 500 2nd instar larvae of root-knot nematodes), BW20 (Rhizoglomus multiployspora CAUBW20 inoculated with 500 2nd instar larvae of root-knot nematodes);

[0016] Figure 5 Figure 2 shows the root node number of tomato root system; CK, RK, PL and BW20 treatments are the same as above.

[0017] Figure 6 Figure 2 shows the nitrogen, phosphorus and potassium absorption of tomato plants; the CK, RK, PL and BW20 treatments are the same as above. DETAILED DESCRIPTION

[0018] The technical solutions in the embodiments of the present invention are described clearly and completely below. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of them. All other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0019] Example 1 Isolation, Identification and Preservation of Rhizoglomus multiployspora CAUBW20

[0020] (1) Separation:

[0021] The soil was collected from Xibeiwang Town, Haidian District, Beijing, at an altitude of 50m and located at (116.117172°E, 40.096806°). After cultivation, the spores were separated by sucrose-wet sieve decantation method, observed under a stereo microscope, and spores of different shapes, sizes, and colors were distinguished. Then, they were inoculated into a blank beaker planted with white clover. After 3-6 months of cultivation, the spores were enriched again by sucrose-wet sieve decantation method, and identified by morphology and molecular biology.

[0022] (2) Identification of morphological characteristics of bacterial species:

[0023] Morphological identification is as important as molecular biological characteristics in the classification of arbuscular mycorrhizal fungi. Morphological identification is based on spore size, color, aggregation mode, number of spore wall layers, Melzer reagent staining characteristics, and hyphae characteristics at the spore connection.

[0024] Depend on Figure 1 Display: The spore aggregation mode of Rhizoglomus multiployspora CAUBW20, which often forms in the root cortex and aggregates into clusters. The clusters are light yellow to brown.

[0025] Spore color: pale yellow to light brown;

[0026] Spore shape and size: spherical or nearly spherical, (28.39-)38.9(-47.28)μm, rarely oval, (31.07-)39.8(-48.01)μm×(23.7-)29.8(-38.9)μm;

[0027] Spore wall: There are three layers of spore wall: SWL1, SWL2, and SWL3; among them: SWL1 characteristics: uniform (without visible sublayers), permanent, semi-flexible, smooth, transparent, thickness of (0.52-)0.8(-1.05)μm, tightly adhered to the SWL2 layer; SWL2 characteristics: permanent, semi-flexible, smooth, light yellow to orange in PLVG solution, thickness of (0.61-)0.69(-0.71), tightly adhered to SWL1, and not separated in broken spores. SWL3 characteristics: uniform, permanent, flexible, easily separated from the lower surface of SWL2 in crushed spores, thickness of (0.5-)0.59(-0.69)μm;

[0028] Spore Melzer reagent (i.e., an aqueous solution of chloral, potassium iodide, and iodine) staining characteristics: SWL1 stains light red in Melzer reagent, and SWL2-3 stains reddish brown;

[0029] Mycelial characteristics: Mycelium is light yellow, straight or curved downward, cylindrical, slightly contracted at the base of the spore, (2.64-)3.30(-3.74)μm wide, the mycelial wall is light yellow to orange, and the mycelial wall is composed of three layers associated with the spore wall. Other characteristics: The spore germination hole is (2.1-)2.34(-2.52)μm wide, open, with no septum below, and the spore contains oily substances.

[0030] The strain of the present invention is different from the arbuscular mycorrhizal fungi reported at home and abroad in morphology, and belongs to a new species that has not been reported yet. The following molecular biological identification obtained the same result.

[0031] (3) Molecular biological identification of strains:

[0032] (1) DNA extraction: The CTAB method was used to extract the DNA of the spores, using the CTAB Plant Genomic DNA Rapid Extraction Kit produced by Beijing Adelaide Company.

[0033] (2) Nested PCR amplification of partial rDNA sequence (SSU-ITS-LSU): Nested PCR amplification of SSU-ITS-LSU and RBP1 sequences in rDNA of the bacterial species was performed. The primers included SSUAmf1, SSUAmf2, SSUCmf1, SSUCmf2, SSUCmf3, LSUAmr1, LSUAmr2, LSUAmr3, LSUA mr4, LSUBmr1, LSUBmr2, LSUBmr3, LSUBmr4, LSUBmr5; RPB1-3F, RPB1-4F, RPB1-5R, RPB1-5RN.

[0034] Based on the sequencing results of the fragments amplified by the above primers, a Bayesian phylogenetic tree and a maximum likelihood phylogenetic tree were constructed.

[0035] The SSU-ITS-LSU phylogenetic tree showed that the three replicate sequencing results of Rhizoglomus multiployspora CAUBW20 of the present invention were clustered into the same branch, and were separated from the other species of Rhizoglomus multiployspora in an independent branch of the genus Rhizoglomus ( Figure 2 ). The support of the Bayesian number and the maximum likelihood number in the phylogenetic tree are 1 and 100, respectively, both of which are maximum. The closest evolutionary species is Rhizoglomus fasciculatim.

[0036] Bayesian phylogenetic tree and maximum likelihood phylogenetic tree of RBP1 sequence Figure 3 As shown. The results of three sequencing of Rhizoglomus multiployspora CAUBW20 strain were also clustered into the same branch, and showed separation from the rest of the species of Rhizoglomus. Unlike the SSU-ITS-LSU phylogeny, the evolutionary relationship of the RBP1 sequence was adjacent to the species Rhizoglomus silesianum and Rhizoglomus irregularis, and the adjacent species of the two sequence evolutionary trees were different. In the phylogenetic trees of SSU-ITS-LSU and RBP1, the sequence identity of Rhizoglomus multiployspora CAUBW20 with the closest species was less than 90.6%. Therefore, the evolutionary relationship of SSU-ITS-LSU and RBP1 sequences showed that Rhizoglomus multiployspora CAUBW20 strain was a new species of Rhizoglomus from the perspective of molecular biology.

[0037] The SSU-ITS-LSU of Rhizoglomus multiployspora CAUBW20 has different morphological characteristics from its related species, Rhizoglomus silesianum. The spore wall of Rhizoglomus multiployspora CAUBW20 is three-layered, while that of Rhizoglomus silesianum is four-layered; the former is light red when stained with SWL1 Melzer reagent, while the latter is light yellow; the spores of Rhizoglomus silesianum are (65-)111(-138)μm; or nearly spherical, 77-80×83-96μm, which is 2.5 times that of Rhizoglomus multiployspora CAUBW20; the hyphae width of Rhizoglomus silesianum is (10.60-)11.8(13.40)μm, which is 3.5 times that of Rhizoglomusmultiployspora CAUBW20.

[0038] In summary, both the molecular biological identification results and the morphological characteristics confirmed that Rhizoglomus multiployspora CAUBW20 was a new species of the genus Rhizoglomus.

[0039] (4) Preservation of strains:

[0040] The strain is deposited in the General Microbiological Center of China Culture Collection Administration, No. 3, No. 1 Beichen West Road, Chaoyang District, Beijing, with the deposit number: CGMCC NO.40588; the deposit date is May 8, 2023. The classification name is Rhizoglomus multiployspora, and the strain number is (CAUBW20). Example 2 Preparation of Rhizoglomus multiployspora CAUBW20 bacterial agent

[0041] Solid medium, liquid medium and sand medium are used, and the number of spores of Rhizoglom usmultiployspora per gram of medium is 70-100. Tomato and carrot are used as host propagation materials, and nutrient soil and sieved river sand (weight ratio 1:4) are used as culture matrix, sterilized at 85° high temperature for 48 hours; 10-50g of pre-expansion bacteria agent is added to 1L of the above sterilized matrix, mixed and plated. The host sowing amount is 10-20 sterilized seeds / pot, and the seeds are sterilized by soaking in 5-8% sodium hypochlorite solution for 10 minutes, and the fungus sand is harvested after 2-4 months of cultivation. The fungus sand contains spores, mycelium and mycorrhizal root segments.

[0042] Example 3 Effect of Rhizoglomus multiployspora CAUBW20 on improving resistance of tomatoes to root knot insects.

[0043] The model variety Little Tom (variety) and the cultivar Hard Powder No. 8 (F1 hybrid) of tomatoes were used, with 4 replicates. 100 spores of Rhizoglomus multiployspora CAUB W20 were added to each pot. On the 7th day after inoculation of Rhizoglomus multiployspora CAUBW20, 500 J2 stage larvae were added to each pot for root knot nematode treatment. The plants were irrigated with modified Hoagland nutrient solution, and the seedlings were harvested on the 40th day after root knot nematode inoculation. Plant biomass, nitrogen, phosphorus, potassium absorption and root knot number were determined. The root knot number was determined by the sodium hypochlorite-acid fuchsin method of root system staining, which was placed in 30 ml of acid glycerol for 1 day for decolorization. The stained root knots were counted under a stereo microscope. SPSS19.0 software was used to statistically process the data. The experimental data were all in x±SE data table. One-way analysis of variance showed that compared with the model group: *P<0.05, **P<0.01, ***P<0.005, ****P<0.001. The results are shown in Table 1. Figure 4 , Figure 5 and Figure 6 .

[0044] Table 1 Biomass of tomato plants under different treatments

[0045]

[0046] Note: CK is blank control, PK is single plant inoculated with 500 2nd instar larvae of root knotworm, PL is Paecilomyces lilacinus + inoculated with 500 2nd instar larvae of root knotworm, BW20 is CAUBW20 inoculated with 500 2nd instar larvae of Rhizoglomus multiployspora. The results are expressed as mean ± standard error (mean ± SE), different letters indicate significant differences at p < 0.05 (Duncan) level (n = 4)

[0047] As shown in Table 1, compared with the control, the inoculation of root knotworms (RK) seriously harmed the accumulation of stem and leaf biomass of tomato (Little Tom), which was reduced from 1.13g / plant in the normal control to 0.75g / plant. After adding the strain of the present invention at the same time, the stem and leaf biomass was 1.49g / plant, even higher than the normal control without inoculation of root knotworms; the root system and the whole plant biomass showed the same trend: the total biomass of the whole plant was 1.21g / plant in the control, 0.82g / plant inoculated with root knotworms, and 1.61g / plant when root knotworms and BW20 were added. Inoculation of this strain completely reversed the growth reduction damage caused by root knotworms, and was even higher than the normal control without inoculation.

[0048] Depend on Figure 4 and Figure 5 The results showed that inoculation with the strain of the present invention effectively increased plant growth, and although the root system was still infected by root knotworms, the biomass was not affected ( Figure 4 ), and the number of root nodes per plant decreased from 36 to 18 ( Figure 5 ).

[0049] Depend on Figure 6 The results show that the nitrogen, phosphorus and potassium absorption of tomato plants significantly reduced the nitrogen, phosphorus and potassium absorption of plants compared with the control RK (inoculated with 500 2nd instar larvae of root knot nematodes), and significantly increased the nitrogen, phosphorus and potassium absorption by inoculating Rhizoglomus multiployspora CAUBW2, which not only reversed the nutrient absorption damage caused by root knot nematodes, but also exceeded the blank control without inoculation of root knot nematodes. According to the research results of this embodiment, the strain of the present invention effectively improves the resistance of tomatoes to root knot nematodes, reduces the damage of root knot nematodes to a level that hardly affects biomass accumulation, and compared with the currently commonly used Paecilomyces lilacinus (PL), the resistance effect is more significant.

[0050] The strain provided by the present invention can not only improve the ability of tomatoes to resist root-knot nematodes, but also has the effect of improving their growth and nutrient absorption. Therefore, it is of great significance to the improvement of tomato production and environmental adaptability. The present invention clarifies the role of this new species of arbuscular mycorrhizal fungi, the polysporic root cyst preparation, in improving the resistance of tomatoes to root-knot nematodes and increasing production, and provides a theoretical basis and technical support for the large-scale promotion and application of the bacterial agent.

[0051] The various embodiments in this specification are described in a progressive manner, and each embodiment focuses on the differences from other embodiments. The same and similar parts between the various embodiments can be referenced to each other.

[0052] With respect to the disclosed above embodiments, it is possible for those skilled in the art to implement or use the present invention. Various modifications to these embodiments will be apparent to those skilled in the art, and the general principles defined herein may be implemented in other embodiments without departing from the spirit or scope of the present invention. Therefore, the present invention will not be limited to the embodiments shown herein, but will conform to the widest scope consistent with the principles and novel features disclosed herein.

Claims

1. A polysporic rhizocystis fungus (Rhizoglomus multiployspora) CAUBW20, characterized by: The polysporic rhizocystis fungus CAUBW20 was deposited in the General Microbiological Center of China Microbiological Culture Collection Administration on May 8, 2023, with a deposit number of CGMCC NO.40588, and the deposit address is No. 3, Yard No. 1, Beichen West Road, Chaoyang District, Beijing, and is classified and named Rhizoglomus multiployspora .

2. The polysporon cysticercus according to claim 1 (Rhizoglomus multiployspora) Application of CAUBW20 in the preparation of bacterial agents for antagonizing root-knot nematodes.

3. The polysporon cysticercus according to claim 1 ( Rhizoglomus multiployspora )Application of CAUBW20 in the preparation of inoculants for improving tomato biomass, yield and nutrient absorption.

4. A bacterial agent that simultaneously antagonizes root-knot nematodes and increases tomato yield and biomass, characterized in that: The bacterial agent includes the polysporon cystis fungus CAUBW20 described in claim 1.

5. The bacterial agent for simultaneously antagonizing root-knot nematodes and increasing tomato yield and biomass according to claim 4, characterized in that: The bacterial agent comprises: a medium, and the medium includes but is not limited to river sand, zeolite, coconut bran, and kaolin.

6. The method for preparing a bacterial agent for simultaneously antagonizing root-knot nematodes and increasing tomato yield and biomass as claimed in claim 5, characterized in that: The process is as follows: nutrient soil and medium are mixed as culture matrix, sterilized at 85°C for 48 hours, and host seeds are sown; then 10-50g of pre-enrichment bacteria agent is added to 1L of the above sterilized matrix, mixed evenly, plated, and cultured.

7. The method for preparing a bacterial agent for simultaneously antagonizing root-knot nematodes and increasing tomato yield and biomass according to claim 6, characterized in that: The host sowing amount is 10-20 sterilized seeds per pot; the host seeds are sterilized before sowing, and the sterilization method is: soaking in 5-8% sodium hypochlorite solution for 10 minutes.

8. The method for preparing a bacterial agent for simultaneously antagonizing root-knot nematodes and increasing tomato yield and biomass according to claim 7, characterized in that: The culture time is 2-4 months, and the bacterial sand is harvested.

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