Prussella sp. ZJUHULYT1 and application thereof in prevention and treatment of gramineae meloidogyne

Abstract

The invention provides a strain of Princisteria sp. ZJUHULYT1 and application thereof in prevention and treatment of Meloidogyne miscaceae, and belongs to the technical field of biological prevention and treatment of Meloidogyne miscaceae, the Princisteria sp. ZJUHULYT1 is preserved in the China Center for Type Culture Collection (CCTCC), and the preservation number is CCTCC NO: M 20251173. The invention further provides a preparation method of the Princisteria sp. ZJUHULYT1 and application of the Princisteria sp. ZJUHULYT1. Although the Princisteria sp. ZJUHULYT1 disclosed by the invention has no direct lethal effect on the gramineous meloidogyne, when the Princisteria sp. ZJUHULYT1 acts on the roots of rice, the number of rice root knots can be remarkably reduced, and invasion of the gramineous meloidogyne can be remarkably inhibited. According to the present invention, the rice root system can be promoted to defend the synthesis of related hormones, the content of 12-oxo-plant dienoic acid (OPDA) and abscisic acid (ABA) in the rice root system can be increased, and the effect of inhibiting the infection of Gramineae Meloidogyne by increasing the hormone content of the rice root can be achieved.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of biological control of root-knot nematodes, and particularly relates to a strain of Pseudomonas sp. ZJUHULYT1 and application thereof in prevention and treatment of Meloidogyne graminicola. BACKGROUND

[0002] Rice is an important food crop in the world, and its yield and quality are related to human food security and life quality. However, the damage of root-knot nematodes has brought great challenges to rice cultivation. Meloidogyne graminicola, commonly known as rice root-knot nematode, is one of the pathogens of rice root-knot nematode disease, and is widely distributed in rice planting areas in Asia, Europe, Africa and America. Rice root system is the direct damage site of Mg, and after infection, the root tip can be induced to be hook-shaped or short rod-shaped, which seriously affects the function of the root system, leading to poor development of rice, and the main symptoms of the aboveground part include growth retardation, leaf discoloration, early flowering, early maturity and less grain.

[0003] At present, the methods for reducing the damage of Meloidogyne graminicola include chemical, physical, agricultural and biological methods, among which the most commonly used is the chemical method. Chemical methods such as bromomethane and metam are chemical fumigation reagents, which have a certain effect, but seriously damage the ozone layer and soil structure; thiazolylphosphine is easy to produce drug damage and has a pungent odor, which is harmful to human health. However, green biological control technologies such as water-flood rotation and biocontrol bacteria have unstable control effects due to unknown defense mechanisms and molecular mechanisms with host plants, and are difficult to completely replace chemical methods in production. With the increasing public awareness of environmental protection and the development needs of green and environmentally friendly agriculture, the limitations of chemical methods such as high pollution, high residue and high toxicity have been widely concerned. At the same time, the use of biocontrol fungi, bacteria and other microbial resources to replace chemical pesticides has become an environmentally friendly nematode control strategy.

[0004] Soil microorganisms, as key soil factors, play an important role in the ecosystem. Soil microorganisms not only participate in the formation of soil structure, the decomposition of organic matter, the transformation of minerals and the fixation of nitrogen, but also have strong anti-nematode ability. Some soil microorganisms can inhibit egg hatching, parasitize nematode bodies, secrete nematocidal secondary metabolites or activate rice immune response to inhibit Mg infection, thereby reducing the number of nematode galls and reproduction. Utilizing soil microorganisms to improve the resistance of rice to Meloidogyne graminicola has the advantages of environmental protection, sustainability and little disturbance to the ecosystem, and is expected to provide a new effective way for the control of rice root-knot nematodes. SUMMARY

[0005] Therefore, the application aims to provide a strain of Pseudomonas ZJUHULYT1 and its application in preventing and treating root-knot nematodes of the family of Gramineae, wherein the Pseudomonas ZJUHULYT1 can inhibit the invasion of the root-knot nematodes of the family of Gramineae by increasing the content of defense hormones in rice roots.

[0006] The application provides a strain of Pseudomonas ZJUHULYT1, which is preserved in the China Center for Type Culture Collection and has a preservation number of CCTCC NO: M 20251173.

[0007] The application provides a biocontrol agent for rice root-knot nematodes, which comprises the Pseudomonas ZJUHULYT1.

[0008] Preferably, the OD of the Pseudomonas ZJUHULYT1 in the biocontrol agent is 0.8-1.2. 600

[0009] The application provides the application of the Pseudomonas ZJUHULYT1 and the biocontrol agent in preventing and treating root-knot nematodes of the family of Gramineae.

[0010] Preferably, the Pseudomonas ZJUHULYT1 is used for root irrigation of rice.

[0011] Preferably, the Pseudomonas ZJUHULYT1 is used in a dosage of 5-15 ml per plant per time, and is used in the seedling stage.

[0012] The application provides the application of the Pseudomonas ZJUHULYT1 and the biocontrol agent in increasing the content of defense hormones in rice roots.

[0013] Preferably, the defense hormones in the rice roots comprise 12-oxo-phytodienoic acid and abscisic acid.

[0014] Compared with the prior art, the application has the following beneficial effects: the application provides a strain of Pseudomonas ZJUHULYT1, which is preserved in the China Center for Type Culture Collection and has a preservation number of CCTCC NO: M 20251173. The Pseudomonas ZJUHULYT1 has no direct effect on root-knot nematodes of the family of Gramineae, but can significantly reduce the number of root knots of rice and inhibit the invasion of the root-knot nematodes of the family of Gramineae when applied to the roots of rice. The Pseudomonas ZJUHULYT1 can promote the synthesis of defense-related hormones in rice roots and increase the content of 12-oxo-phytodienoic acid (OPDA) and abscisic acid (ABA) in the rice roots, indicating that the Pseudomonas ZJUHULYT1 can inhibit the invasion of the root-knot nematodes of the family of Gramineae by increasing the content of hormones in rice roots.​ BRIEF DESCRIPTION OF DRAWINGS

[0015] Figure 1 A colony morphology chart of the Priestia sp. ZJUHULYT1;

[0016] Figure 2 A phylogenetic tree of the Priestia sp. ZJUHULYT1;

[0017] Figure 3 A survival rate of the nematode after the bacterial solution of the Priestia sp. ZJUHULYT1 is treated for 24 hours;

[0018] Figure 4 An effect of the bacterial solution of the Priestia sp. ZJUHULYT1 on the number of rice root nodules;

[0019] Figure 5 An effect of the bacterial solution of the Priestia sp. ZJUHULYT1 on the content of defense hormones in the rice roots.

[0020] BIOLOGICAL DEPOSIT DESCRIPTION

[0021] The Priestia sp. ZJUHULYT1 is preserved in the China Center for Type Culture Collection, the preservation address is Wuhan University, Wuhan, China, the preservation date is May 26, 2025, and the preservation number is CCTCC NO: M20251173. DETAILED DESCRIPTION

[0022] The present application provides a Priestia sp. ZJUHULYT1, which is preserved in the China Center for Type Culture Collection, and the preservation number is CCTCC NO: M20251173.

[0023] The Priestia sp. ZJUHULYT1 provided in the present application is isolated and purified from the soil of the rice roots infected by the Paratrichodorus terrestroides; the Priestia sp. ZJUHULYT1 has the following biological characteristics: after being cultured on a TSA plate at 30 DEG C for 1 day, the morphological observation result of the strain shows that the colony is round, slightly yellow, the surface is flat and moist, the inside of the colony is mucilaginous, the diameter is about 2.0-5.0 mm, and the gram reaction is positive. The present application inoculates the strain ZJUHULYT1 into the rice, and significantly improves the resistance of the rice to the Paratrichodorus terrestroides.

[0024] The present application also provides a biocontrol agent for the rice root nematode, which comprises the Priestia sp. ZJUHULYT1. In the present application, the OD 600is 0.8-1.2, preferably 0.9-1.1, and more preferably 1.0. The form of the biocontrol agent is not particularly limited in the present application, and either a liquid bacterial agent or a solid bacterial agent can be used.

[0025] The present application also provides the application of the Lysobacter ZJUHULYT1 and the biocontrol agent in the prevention and treatment of the root-knot nematode of the graminaceous plants.

[0026] In the present application, the rice is preferably irrigated with the Lysobacter ZJUHULYT1; the amount of the Lysobacter ZJUHULYT1 used is preferably 5-15 ml / plant / time, and more preferably 8-12 ml / plant / time; the application time of the Lysobacter ZJUHULYT1 is not particularly limited in the present application, and the Lysobacter ZJUHULYT1 is preferably used in the seedling stage.

[0027] The present application also provides the application of the Lysobacter ZJUHULYT1 and the biocontrol agent in the increase of the content of the root defense hormones of the rice. In the present application, the root defense hormones of the rice preferably include 12-oxo-phytodienoic acid and abscisic acid. After the rice is irrigated with the Lysobacter ZJUHULYT1, the content of 12-oxo-phytodienoic acid (OPDA) and abscisic acid (ABA) in the rice root system is significantly increased, which indicates that the Lysobacter ZJUHULYT1 can inhibit the infection of the root-knot nematode of the graminaceous plants by increasing the content of the hormones in the rice root.

[0028] The technical solutions provided by the present application are described in detail below in combination with the examples, but they should not be understood as limitations to the protection scope of the present application.

[0029] Example 1

[0030] Isolation and identification of the Lysobacter ZJUHULYT1

[0031] 1.1 Isolation of the Lysobacter ZJUHULYT1

[0032] (1) Acquisition of the rhizosphere soil: in the vigorous growth period of the rice, the rice roots at a depth of 0-10 cm were collected, the relatively loose particles on the rice roots were shaken off, and then the rice roots were put into a 50 mL centrifuge tube containing 25 mL of PBS buffer, and oscillated at 2000 rpm for 1 min. The oscillated roots were taken out and put into a new 50 mL centrifuge tube containing 25 mL of PBS buffer, and oscillated at 2000 rpm for 1 min. After the roots were taken out, the two PBS were combined, and centrifuged at 5000 rpm and 4℃ for 5 min to remove the supernatant to obtain the rhizosphere soil of the rice, which was stored at -80℃ for standby.

[0033] (2) Rhizosphere soil single colony acquisition: 1 g of rhizosphere soil was weighed into a 50 mL sterile centrifuge tube, 10 mL of sterile water was added, vortexed evenly, and then gradient dilution was performed to obtain 10 -6 ,10 -7 ,10 -8 gradient soil suspensions. 30 μL of soil suspension from each gradient was taken and evenly coated on different culture medium plates using a coating rod, LB, R2A, TSA, 1 / 10 LB, 1 / 10 R2A, 1 / 10 TSA, and the growth conditions of the plates were observed. Single colonies were picked and purified using the corresponding medium, and the purified single colonies were obtained (results shown in Figure 1 .

[0034] 1.2 Identification of Pseudomonas ZJUHULYT1

[0035] The single colonies obtained in the above step were dissolved in a PCR tube containing 100 μL of sterile water and incubated at 95°C for 10 min in a PCR instrument as a template. The template was subjected to PCR amplification using 16S full-length primers 27F and 1492R.

[0036] The PCR reaction used a Takara primer star kit (purchased from Baorui Biotechnology (Beijing) Co., Ltd.), and the PCR conditions were as follows: 98°C pre-denaturation for 45 s, 98°C denaturation for 10 s, 60°C annealing for 5 s, 72°C extension for 2 min, 31 cycles, and 72°C extension for 2 min. The PCR amplification product was sent to Zhejiang Shangya Biotechnology Co., Ltd. for sequencing. The sequencing results showed that the 16sRNA sequence was 1454 bp long, and the nucleotide sequence was as follows (SEQ ID NO. 1):

[0037]

[0038] The 16S rRNA sequence obtained above was subjected to comparative analysis using the NCBI blast function, and a phylogenetic tree was constructed using MEGA12 software in combination with the blast results and the corresponding model species to determine the taxonomic status. The results are shown in Figure 2 The above analysis determined that strain ZJUHULYT1 is a Priestia. Morphological observation of the strain showed that the colony was round, slightly yellow, with a flat and moist surface, and the inside of the colony was mucilaginous, with a diameter of about 2.0-5.0 mm. The Gram reaction was positive.

[0039] Example 2

[0040] Direct effect of Priestia ZJUHULYT1 on nematodes

[0041] 2.1 A loop of bacteria was picked up from the bacteria solution in the cryopreservation tube containing 30% glycerol, inoculated into a shaking tube containing 4 mL of TSB liquid medium, and cultured in a 30°C incubator at 200 rpm for 24 h until the OD 600 value of the bacteria solution was 1.5; the bacteria solution after shaking was centrifuged at 5000 rpm for 5 min, washed twice with PBS suspension after removing the supernatant, and washed 3 times with sterile water to adjust the OD 600 of the bacteria solution to 1.0, and a bacteria suspension was obtained.

[0042] 2.2 The infected rice roots of Meloidogyne graminicola were cut into pieces, placed in a collection device, and incubated at 28°C to collect fresh hatching J2s of Meloidogyne graminicola, which were collected once a day and stored at 4°C for later use; the collected nematodes were soaked in a solution containing 200 mg / L streptomycin sulfate for 4 h, and then washed with sterile 0.085% NaCl to obtain sterile nematodes.

[0043] 2.3 The sterile nematodes were dissolved in sterile water and adjusted to a concentration of 1000 heads / mL to obtain a nematode suspension; 50 μL of the nematode suspension was taken into a 96-well plate, and 50 μL of sterile water / bacteria suspension (OD 600 = 1) was added to obtain a sterile water treatment group / bacteria suspension treatment group. The number of dead nematodes was observed using an inverted phase contrast microscope at 0 h and 24 h after the addition was completed, and the survival rate of the nematodes after 24 h was calculated. Survival rate (%) = number of surviving nematodes / total number of nematodes x 100

[0044] Corrected mortality rate (%) = (treatment group mortality rate - control group mortality rate) / (1 - control group mortality rate) x 100

[0045] The results are shown in Table 1.Figure 3 The results show that the strain ZJUHULYT1 has no direct lethal effect on the root-knot nematode within 24h.

[0046] Example 3

[0047] Effect of ZJUHULYT1 on rice against root-knot nematode

[0048] The rice variety used for the experiment was Nipponbare. The rice seeds were placed in a 30°C light incubator for 10 days for germination, and the water was changed once a day. After 10 days of germination, the rice was transplanted into Hoagland's nutrient solution culture boxes and placed in a greenhouse. The light and dark cycle was 14h / 10h, the temperature was 28°C / 25°C, and the humidity was about 60%. After the rice grew to the seedling stage, the rice was transplanted into black flowerpots (10 cm x 10 cm x 20 cm) filled with sand in advance. The rice was watered and fed with nutrient solution as needed. After the rice grew for 14 days, 10 mL of bacterial suspension of strain ZJUHULYT1 (OD 600 = 1.0) was used for root irrigation treatment. After 48h of root irrigation, holes were punched around the rhizosphere soil of each rice plant, and 200 root-knot nematodes J2s were inoculated per pot. After 7 days of inoculation, the rice roots were collected to obtain the number of root knots. A control group (H2O) was set up by applying the same volume of sterile water. The results are shown in Figure 4 The results show that the number of root knots in the treatment group (bacterial suspension) with strain ZJUHULYT1 added was reduced by 35.7% compared with the control group (CK) with H2O, which can significantly inhibit the invasion of root-knot nematodes.

[0049] Example 4

[0050] Effect of ZJUHULYT1 on rice root hormones

[0051] According to the method of Example 3, the rice roots were collected in liquid nitrogen 24h after inoculation with the nematode suspension, and the samples were stored in a -80°C refrigerator. The hormones in the roots of rice with and without bacterial suspension were extracted and determined. The specific method of hormone determination is as follows:

[0052] (1) Materials and reagents

[0053] Plant hormone extraction solution: 990μl of ethyl acetate and 10μl of isotope-labeled hormone standard mixture were mixed to prepare 1mL of hormone extraction solution; the isotope-labeled hormone standard mixture was 100ng / mL of d6-JA, d6-ABA, d4-SA, d6-JA-Ile and d5-IAA. 70% methanol solution: prepared by mixing methanol and deionized water in a volume ratio of 70:30;

[0054] (2) Extraction step

[0055] ① The collected rice root system was rapidly frozen in liquid nitrogen, and then ground into fine powder in a liquid nitrogen environment. 0.100±0.050 g of the sample was weighed into a 2 mL round-bottom centrifuge tube and the sample weight was recorded. The sample was stored in a-80℃ refrigerator;

[0056] ② 1 mL of plant hormone extraction solution was added to the centrifuge tube, and the sample was quickly and thoroughly vortexed on a vortex instrument to ensure that the extraction solution fully wrapped the sample particles for protection;

[0057] ③ The centrifuge tube was placed on a vortex shaker and vortexed for another 10 min. Then the centrifuge tube was transferred to a table centrifuge, and centrifuged at 12000 rpm for 15 min at 4℃ to prevent sample degradation;

[0058] ④ The supernatant was transferred to a new 2 mL round-bottom centrifuge tube, and then the centrifuge tube was opened and placed in a table vacuum centrifugal concentrator, and concentrated at 30℃ for 32-35 min until the ethyl acetate in the tube was just evaporated;

[0059] ⑤ 150 μL of 70% methanol solution was added to the centrifuge tube, and vortexed on a vortex shaker for 5 min;

[0060] ⑥ The centrifuge tube was transferred to a table centrifuge, and centrifuged at 13000 rpm for 10 min at 4℃;

[0061] ⑦ 100 μL of the supernatant was transferred to a sample bottle with an inner cannula using a syringe, and the label was written.

[0062] (3) Machine analysis: the sample was analyzed by UPLC-QQQ-MS / MS system

[0063] The results are shown in Figure 5 It is shown that the treatment group (inoculation) added with the bacterial suspension of strain ZJUHULYT1 and the control group (CK) added with H2O, after 24 h of adding the nematode suspension, the treatment group added with strain ZJUHULYT1 can promote the synthesis of defense-related hormones in rice root system, and the contents of 12-oxo-phytodienoic acid (OPDA) and abscisic acid (ABA) in the rice root system inoculated with strain ZJUHULYT1 are significantly increased, indicating that ZJUHULYT1 can inhibit the infection of paradriftia root knot nematode by increasing the hormone content of rice root.

[0064] From the above examples, it can be known that the priestly bacteria ZJUHULYT1 can promote the synthesis of defense-related hormones in rice root system, and then increase the resistance of rice by increasing the hormone content of rice root, and then achieve the effect of inhibiting the infection of paradriftia root knot nematode.

[0065] The above merely preferred embodiments of the present application, it should be noted that for those of ordinary skill in the art, without departing from the principles of the present application, can also make a number of improvements and refinements, these improvements and refinements should also be considered within the scope of the present application.

Claims

1. A strain of Priestia sp. ZJUHULYT1, characterized in that: It is deposited in China Center for Type Culture Collection with the deposit number CCTCC NO: M 20251173.

2. A biocontrol agent for rice root-knot nematodes, characterized in that: The invention comprises the Priesteria sp. ZJUHULYT1 described in claim 1.

3. The biocontrol agent according to claim 2, characterized in that The OD of Priesteria ZJUHULYT1 in the biocontrol agent 600 It is 0.8 to 1.

2.

4. Use of the Priesteria ZJUHULYT1 according to claim 1 and the biocontrol agent according to claim 2 or 3 in controlling root-knot nematodes of the Poaceae family.

5. The use according to claim 4, characterized in that The Priesteria ZJUHULYT1 was used to irrigate the roots of rice.

6. The use according to claim 5, characterized in that The dosage of the Priesteria sp. ZJUHULYT1 is 5-15 ml / plant / time; the Priesteria sp. ZJUHULYT1 is used during the seedling stage.

7. Use of the Priesteria ZJUHULYT1 according to claim 1 and the biocontrol agent according to claim 2 or 3 in increasing the content of defense hormones in rice roots.

8. The use according to claim 7, characterized in that The rice root defense hormones include 12-oxophytodienoic acid and abscisic acid.

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