Bacillus ZJUHUSRH1 and application thereof in prevention and control of pseudocereal meloidogyne

Abstract

The invention provides bacillus ZJUHUSRH1 and application thereof in prevention and control of pseudocereal meloidogyne, and belongs to the technical field of biological prevention and control of meloidogyne, the bacillus ZJUHUSRH1 is preserved in the China Center for Type Culture Collection, the preservation number is CCTCC NO: M 20251183, the preservation number is CCTCC NO: M 20251183, and the preservation number is CCTCC NO: M 20251183. The bacillus ZJUHUSRH1 has a remarkable prevention and treatment effect on rice pseudocereal meloidogyne in soil and can effectively prevent and treat infection and reproduction of the rice meloidogyne; meanwhile, the bacillus ZJUHUSRH1 can increase the hormone content of the rice root by activating a salicylic acid synthesis pathway, so that the effect of inhibiting invasion of the pseudocereal meloidogyne is achieved.

Description

Technical Field

[0001] The invention belongs to the technical field of biological control of root-knot nematodes, and in particular relates to a Bacillus ZJUHUSRH1 strain and an application thereof in controlling root-knot nematodes of cereals. Background Art

[0002] In recent years, rice cultivation has continued to expand. However, with the prevalence of intensive farming practices, soil-borne rice diseases have become increasingly severe. Rhizosphere diseases caused by plant-parasitic nematodes have become a key obstacle to stable rice yields. Over 300 plant-parasitic nematode species have been found to infect and damage rice roots. Among them, the root-knot nematode (Meloidogyne graminicola) is the most prominent due to its unique pathogenic mechanism and rapid reproduction. This nematode has a life cycle of only 20 to 27 days, progressing through the egg, first-instar larvae (J1s), second-instar infective larvae (J2s), third-instar (J3s), fourth-instar larvae (J4s), and adult stages. After invading the rice root apex, J2s induce cellular reprogramming to form giant cells that serve as a nutrient source. This leads to root malformations and impedes nutrient absorption, ultimately resulting in reduced yield and quality.

[0003] Currently, control of the rice root-knot nematode (Meloidogyne graminicola) primarily relies on chemical pesticides (such as thiazolyl) and resistant varieties, supplemented by agricultural practices (such as crop rotation and flooding) and biological control (such as Paecilomyces lilacinus). Chemical control is the most widely used due to its rapid effectiveness. However, chemical pesticides can harm the environment and the ecological balance of species. Residues can enter the human body through a series of ecosystem cycles, food chains, and food webs, potentially harming human health. Biological control agents offer advantages over chemical agents, such as high targeting, easy degradation, and reduced environmental pollution. Therefore, they may be considered an ideal alternative to chemical pesticides in the future, offering promising development prospects and broad-spectrum application value.

[0004] Rhizosphere microorganisms play an important role in maintaining plant health and soil ecological functions. They also have a certain control effect on the root-knot nematode disease of rice. Rhizosphere microorganisms have advantages such as strong colonization ability and strong stress resistance, making them an important source for the development of biopesticides. However, current nematicide formulations based on rhizosphere microorganisms are still in their early stages. There is an urgent need to overcome key technical bottlenecks and develop new and highly effective biological nematicides, which are of great significance to ensuring food production safety. Summary of the Invention

[0005] In view of this, the object of the present invention is to provide a Bacillus ZJUHUSRH1 and its application in controlling root-knot nematodes of rice; the Bacillus ZJUHUSRH1 has a significant control effect on root-knot nematodes of rice in the soil, and can effectively control the infection and reproduction of root-knot nematodes of rice.

[0006] The invention provides a Bacillus ZJUHUSRH1, which is deposited in China Center for Type Culture Collection with a deposit number of CCTCC NO: M 20251183.

[0007] The invention provides a biocontrol agent, which comprises the Bacillus ZJUHUSRH1.

[0008] Preferably, the biocontrol agent is a liquid agent or a solid agent, and the concentration of Bacillus ZJUHUSRH1 in the biocontrol agent is 1×10 5 ~1×10 9 CFU / mL.

[0009] The present invention provides a method for controlling rice root-knot nematodes by utilizing the Bacillus ZJUHUSRH1 and the biocontrol agent, wherein the Bacillus ZJUHUSRH1 or the biocontrol agent is used to act on rice.

[0010] Preferably, the Bacillus ZJUHUSRH1 is applied during the seedling stage, the number of applications is 1 to 3 times, and the interval between two adjacent applications is 7 to 10 days.

[0011] Preferably, the application amount of the Bacillus ZJUHUSRH1 is 4 to 6 ml / strain / time.

[0012] Preferably, the application method of the Bacillus ZJUHUSRH1 includes spraying, broadcasting or soil mixing.

[0013] The present invention provides the application of the Bacillus ZJUHUSRH1 and the biocontrol agent in improving the hormone synthesis in rice roots.

[0014] Preferably, the rice root hormones include salicylic acid and 12-oxo-phytodienoic acid.

[0015] Compared with the prior art, the present invention has the following beneficial effects: the present invention provides a Bacillus ZJUHUSRH1, which is isolated from rice rhizosphere soil. The Bacillus ZJUHUSRH1 has a significant control effect on rice root-knot nematodes in the soil and can effectively control the infection and reproduction of rice root-knot nematodes; at the same time, the Bacillus ZJUHUSRH1 can also regulate the content of plant hormones in rice roots, significantly increase the content of salicylic acid and 12-oxo-phytodienoic acid, and reduce jasmonic acid and jasmonic acid-isoleucine complexes; the Bacillus ZJUHUSRH1 can increase the hormone content in rice roots by activating the salicylic acid synthesis pathway, thereby achieving the effect of inhibiting the invasion of the rice root-knot nematodes. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] Figure 1 This is the plate colony observation result of Bacillus ZJUHUSRH1;

[0017] Figure 2 This is the phylogenetic analysis result of Bacillus ZJUHUSRH1;

[0018] Figure 3 The root knot number of rice roots infected by M. graminearum was measured after application of Bacillus ZJUHUSRH1;

[0019] Figure 4 is the dry weight of underground rice roots infected by M. graminearum after application of Bacillus ZJUHUSRH1;

[0020] Figure 5 The change in hormone content in rice infected with the root-knot nematode genus M. graminearum after application of Bacillus ZJUHUSRH1 was compared with that in the control rice without application of the strain.

[0021] Biological Deposit Description

[0022] Bacillus sp. ZJUHUSRH1 was deposited in the China Center for Type Culture Collection, Wuhan University, Wuhan, China. The deposit date is May 26, 2025, and the deposit number is CCTCC NO: M 20251183. DETAILED DESCRIPTION

[0023] The invention provides a Bacillus ZJUHUSRH1, which is deposited in China Center for Type Culture Collection with a deposit number of CCTCC NO: M 20251183.

[0024] In the present invention, the Bacillus ZJUHUSRH1 is isolated from rice rhizosphere soil. The colonies of the Bacillus ZJUHUSRH1 are light yellow, opaque, with a rough and dry surface, no halo, regular edges, Gram-positive staining, and a colony size of 0.5 to 1.0 mm.

[0025] The present invention provides a biocontrol agent, which comprises the Bacillus ZJUHUSRH1. In the present invention, the biocontrol agent is preferably a liquid agent or a solid agent, and the concentration of Bacillus ZJUHUSRH1 in the biocontrol agent is 1×10 5 ~1×10 9 CFU / mL, preferably 1×10 7 ~1×10 8 In the present invention, when the biological agent is a liquid agent, it is preferably obtained by suspending the bacteria in sterile water, PBS buffer or culture medium.

[0026] The present invention also provides a method for controlling rice root-knot nematodes by using the Bacillus ZJUHUSRH1 and the biocontrol agent, wherein the Bacillus ZJUHUSRH1 or the biocontrol agent is used to act on rice.

[0027] In the present invention, the application period of the Bacillus ZJUHUSRH1 is preferably the seedling stage, the number of applications is preferably 1 to 3 times, and the interval between two adjacent applications is preferably 7 to 10 days. The application amount of the Bacillus ZJUHUSRH1 is preferably 4 to 6 ml / plant / time, more preferably 4.5 to 5.5 ml / plant / time; the application method of the Bacillus ZJUHUSRH1 includes spraying, broadcasting or soil mixing. In the specific implementation process of the present invention, the rice is planted in a culture pot, and the specifications of the culture pot are preferably 8 to 12×8 to 12×16 to 24 cm, more preferably 9 to 11×9 to 11×18 to 22 cm, and even more preferably 10×10×20 cm.

[0028] The present invention provides the use of the Bacillus sp. ZJUHUSRH1 and the biocontrol agent for increasing hormone synthesis in rice roots. In the present invention, the rice root hormones include salicylic acid and 12-oxo-phytodienoic acid. Treatment with Bacillus sp. ZJUHUSRH1 has no significant effect on the levels of indoleacetic acid (IAA) and abscisic acid (ABA) in rice roots, but significantly increases the levels of salicylic acid (SA) and 12-oxo-phytodienoic acid (OPDA), and decreases the levels of jasmonic acid (JA) and jasmonic acid-isoleucine (JA-Ile).

[0029] The technical solutions provided by the present invention are described in detail below with reference to the embodiments, but they should not be construed as limiting the scope of protection of the present invention.

[0030] Example 1

[0031] During the vigorous growth period of rice, rice roots at a depth of 0 to 10 cm were collected: after shaking off the loose particles on the rice roots, they were placed in a 50 mL centrifuge tube containing 25 mL of PBS buffer and vortexed for 15 seconds using a vortexer. The vortexed roots were taken out and placed in a new 50 mL centrifuge tube containing 25 mL of PBS buffer and vortexed for 15 seconds using a vortexer. After taking out the roots, the PBS was combined twice and centrifuged at 8000 rpm and 4°C for 5 minutes to obtain the rhizosphere soil.

[0032] Weigh 1g of rhizosphere soil and place it in a 50mL sterile test tube. Add 10mL of sterile water, vortex evenly, and then dilute it in series to obtain 10 -6 , 10 -7 , 10 -8 , 10 -9 Gradient soil suspension. Take 100 μL of each of the four gradient soil suspensions and spread them evenly on LB, 1 / 10 LB, R2A, 1 / 10 TSA, TSA, and AXB plates using a spreading rod. Observe the growth status of the plates within 24 to 120 hours (the results are shown in the figure). Figure 1 Pick a single colony, and use the corresponding culture medium for streak purification to obtain a purified single colony.

[0033] A single colony after purification was picked, dissolved in a PCR tube containing 100 μL of sterile water, and incubated at 98°C for 5 minutes in a PCR instrument as a template; the template was PCR amplified using the 16S full-length primers 27F, 1492R.

[0034] PCR reactions were performed using the Takara Primestar kit (purchased from Bio-Tech (Beijing) Co., Ltd.). PCR conditions were as follows: 98°C pre-denaturation for 45 seconds, followed by 31 cycles of denaturation at 98°C for 10 seconds, annealing at 60°C for 5 seconds, and extension at 72°C for 2 minutes, followed by extension at 72°C for 2 minutes. The PCR amplification products were purified using 1% agarose gel electrophoresis and sent to Zhejiang Shangya Biotechnology Co., Ltd. for sequencing.

[0035] The sequencing results showed that the 16S rRNA sequence amplified from strain ZJUHUSRH1 was 1454 bp long, and its nucleotide sequence was shown in SEQ ID NO.1:

[0036]

[0037] The 16S rRNA sequences obtained above were compared and analyzed using NCBI blast, and the blast results were combined with the corresponding model species for phylogenetic analysis to obtain the identification results. Figure 2 As shown, through the above analysis, the strain ZJUHUSRH1 was determined to be Bacillus.

[0038] The morphological observation results of the strain showed that the colonies were light yellow, opaque, with a rough and dry surface, no halo, regular edges, Gram-positive, and the colony size was 0.5-1.0 mm.

[0039] Example 2

[0040] The shallow tray method is used to isolate R. graminearum. Specifically, the shallow tray separation device consists of two plastic shallow trays, one with a coarse mesh bottom, placed on the other tray. Gauze is gently placed into the tray, ideally with water just covering the surface of the tray. Roots of Nipponbare rice (a variety of rice specifically cultivated for nematode reproduction) infected with R. graminearum are selected, chopped, and placed on the gauze on the sieve tray. Purified water is then slowly poured in to submerge the sample. After 7 days of incubation, the sample is sterilized by immersion in a solution containing 200 mg / L streptomycin sulfate, 25 mg / L rifampicin, and 1× CellGuard (purchased from Jiangsu Jirui Biotechnology Co., Ltd., Catalog No. Gp5507) for 4 hours. The sterilized root-knot nematodes are then washed with sterile 0.085% NaCl to obtain sterile nematodes.

[0041] After the suspension was stirred evenly, the number of rice root-knot nematodes in each 50 μL of liquid was observed under a microscope, and the number of nematodes in the total liquid was calculated. It was appropriate that 1 ml of solution contained 200 J2 nematodes.

[0042] In the clean bench, an inoculating loop was used to pick up a loop of Bacillus ZJUHUSRH1 bacterial solution stored in a 30% glycerol cryopreservation tube and inoculated into a shaking bacterial tube containing LB liquid culture medium (purchased from Beijing Solebow Technology Co., Ltd., product number L8291). In a 30°C incubator, Bacillus ZJUHUSRH1 was shaken at 180 rpm for 24 hours until the OD value of the bacterial solution was between 1.0 and 1.5; the shaken bacterial solution was centrifuged at 6000 rpm for 2 minutes, the supernatant was collected, and the bacteria were treated by resuspension centrifugation. After removing the supernatant, the cells were washed twice with PBS resuspension solution and washed once with sterile water, and the bacterial solution OD was adjusted to 1.0 to obtain a bacterial suspension.

[0043] Example 3

[0044] Before germination, Nipponbare rice seeds were disinfected by soaking them in a 1 / 1000 concentration of prochloraz and imidacloprid (purchased from Dow AgroSciences (China) Co., Ltd.). Germination was then carried out in a 30°C light incubator for 10 days, with the water changed daily. Simultaneously, river sand was prepared for autoclaving. After germination, the rice seedlings were planted in 10 × 10 × 20 cm pots using the sterilized river sand.

[0045] When the rice plants grew to the seedling stage, a treatment group (SRH1+MG) and a control group (MG) were set up, with 12 replicates each. Each treatment group was inoculated with 5 mL of the bacterial suspension (10 8 CFU / ml), and 5 ml of sterile water was added to each control group.

[0046] 21 days after rice planting, each pot of rice was inoculated with 200 sterile root-knot nematodes J2 obtained by the method of Example 2. 7 days after inoculation, the rice roots were collected to determine the number of rice root knots.

[0047] The rice roots were cut off and dried in an oven at 60°C for 48 h, and then their dry weight was weighed to obtain the rice biomass.

[0048] Rice root knot number Figure 3 As shown in the results, Bacillus ZJUHUSRH1 can significantly reduce the infection degree of M. graminearum on rice roots.

[0049] Rice root dry weight Figure 4 As shown, Bacillus ZJUHUSRH1 had no significant growth-promoting effect, but also had no negative impact on rice growth.

[0050] Experimental Example 4

[0051] According to the method in Example 3, rice was planted and treated with nematodes and bacterial solution. 12 replicates were set for each of the treatment group (SRH1+MG) and the control group (MG). The rice roots were collected, rapidly frozen in liquid nitrogen for 30 minutes, and ground to obtain rice root powder. An internal standard solution was prepared in advance. The internal standard solution used 100% methanol as the solvent. The final internal standard solution contained deuterated jasmonic acid-D5 (d5-JA), carbon 13 labeled jasmonic acid-isoleucine ( 13 The samples were mixed with 100 ng / mL of each of deuterated salicylic acid (d4-SA), deuterated abscisic acid (d6-ABA), and deuterated indoleacetic acid (d5-IAA). A standard mix was then prepared, containing 990 μL of ethyl acetate as the extract and 10 μL of internal standard per 1 mL.

[0052] Weigh 0.1g of rice root powder, add 1mL of mixed standard to every 0.1g of rice root powder, vortex on a vortex instrument for 10min, centrifuge at 4℃ and 12000rpm for 20min, draw the supernatant into a new 2mL centrifuge tube, and evaporate on a rotary evaporator for 35min to concentrate the plant hormones; finally, add 200μL of 70% methanol to each concentrated centrifuge tube, vortex for 5min, centrifuge at 4℃ and 13000rpm for 10min, draw 100μL of supernatant into a sample bottle (2mL) with an inner tube (250μL) placed to extract the plant hormones in the rice root powder, and analyze it by UHPLC-MS / MS to obtain the hormone content in the rice with different treatments. The results are as follows Figure 5 shown.

[0053] The results showed that treatment with strain ZJUHUSRH1 could promote the synthesis of defense-related hormones in rice roots; and compared with the control group, the contents of salicylic acid and 12-oxo-phytodienoic acid in rice roots treated with strain ZJUHUSRH1 were significantly increased, and the contents of jasmonic acid and jasmonic acid-isoleucine complex decreased compared with those without strain treatment. It is known that the salicylic acid pathway has a certain inhibitory effect on the jasmonic acid pathway, which further proves that strain ZJUHUSRH1 can increase the hormone content in rice roots by activating the salicylic acid synthesis pathway to achieve the effect of inhibiting the invasion of root-knot nematodes.

[0054] As demonstrated in the above examples, the Bacillus sp. ZJUHUSRH1 provided by the present invention has a significant control effect on the root-knot nematode of rice in the soil. Directly treating rice plants with a bacterial solution of this strain effectively prevents the infection and reproduction of the root-knot nematode. Furthermore, this strain can enhance plant resistance by regulating the levels of plant hormones in rice. This present invention provides a new method for the green control of the root-knot nematode of rice.

[0055] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principles of the present invention. These improvements and modifications should also be regarded as within the scope of protection of the present invention.

Claims

1. A Bacillus sp. ZJUHUSRH1, characterized in that: It is deposited in China Center for Type Culture Collection with the deposit number CCTCC NO: M 20251183.

2. A biocontrol agent, characterized in that The biocontrol agent comprises the Bacillus ZJUHUSRH1 according to claim 1.

3. The biocontrol agent according to claim 2, characterized in that The biocontrol agent is a liquid agent or a solid agent, and the concentration of Bacillus ZJUHUSRH1 in the biocontrol agent is 1×10 5 ~1×10 9 CFU / mL.

4. A method for controlling root-knot nematodes of rice using the Bacillus ZJUHUSRH1 according to claim 1 and the biocontrol agent according to claim 2 or 3, characterized in that: The Bacillus ZJUHUSRH1 or the biocontrol agent is used to act on rice.

5. The method according to claim 4, characterized in that The application period of the Bacillus ZJUHUSRH1 is the seedling stage, the application frequency is 1 to 3 times, and the interval between two adjacent applications is 7 to 10 days.

6. The method according to claim 5, characterized in that The application amount of the Bacillus ZJUHUSRH1 is 4 to 6 ml / strain / time.

7. The method according to any one of claims 4 to 6, characterized in that The application method of the Bacillus ZJUHUSRH1 includes spraying, broadcasting or soil mixing.

8. Use of the Bacillus ZJUHUSRH1 according to claim 1 and the biocontrol agent according to claim 2 or 3 in improving hormone synthesis in rice roots.

9. The use according to claim 8, characterized in that The rice root hormones include salicylic acid and 12-oxo-phytodienoic acid.

Images