Bacillus velezensis ZJUHUCC1 and application thereof

Abstract

The invention provides bacillus velezensis ZJUHUCC1 and application thereof, and belongs to the technical field of agricultural microorganisms, the preservation number of the bacillus velezensis ZJUHUCC1 is CCTCC (China Center for Type Culture Collection) NO: M 2025095, and the preservation number of the bacillus velezensis ZJUHUCC1 is CCTCC NO: M 2025095. The invention further provides a microbial agent, and the microbial agent comprises the bacillus velezensis ZJUHUCC1 and the bacillus velezensis ZJUHUCC1. The viable bacterium concentration of the bacillus velezensis ZJUHUCC1 in the microbial agent is 1 * 10 < 5 > to 1 * 10 < 7 > CFU / mL. The bacillus velezensis ZJUHUCC1 disclosed by the invention can obviously promote the growth of corn crops, improve the sex hormone resistance and system resistance level of corn and reduce the capability of infecting the corn by meloidogyne incognita.

Description

Technical Field

[0001] The invention belongs to the technical field of agricultural microorganisms, and in particular relates to a strain of Bacillus velez ZJUHUCC1 and an application thereof. Background Art

[0002] Corn is one of the most widely grown food crops in the world and plays an important role in global food security. Its unique growth characteristics, nutritional value and economic significance make corn an indispensable part of agricultural crops.

[0003] Due to the influence of multiple factors such as continuous crop planting, soil-borne nematode diseases have become a common disease of corn. Among them, the southern root-knot nematode (Meloidogyne incognita) is an important pathogen of corn. After infecting corn, it will inhibit the growth and development of the corn root system. In severe cases, it will cause the death of corn and result in corn crop failure. Southern root-knot nematodes parasitize on the roots of plants. Adults and larvae will form tumor-like structures in the root system, resulting in stunted root growth and affecting the plant's nutrient and water absorption. Southern root-knot nematodes have a short life cycle, usually 28 to 40 days, with a fast reproduction rate and strong adaptability, which allows them to spread and spread rapidly and survive in a variety of soil conditions. Infected plants often show slow growth, dwarfing, yellowing leaves, and may eventually lead to death.

[0004] At present, the most commonly used prevention and control measure for nematodes is chemical control, using nematode pesticides for soil treatment, but many chemical control methods seriously pollute water, air and soil environments, posing a serious threat to human health and ecological balance. Therefore, the development and adoption of environmentally friendly biological methods has sustainable development prospects. One of the new technologies of biological control is to separate and screen microbial agents that are beneficial to plants, and to find microorganisms that have the effects of promoting plant growth, increasing crop yields, and inducing plants to produce systemic resistance (Induced systemic resistance, ISR), thereby improving the overall resistance of plants to diseases and pests. Therefore, the development of green and friendly functional microbial agents will show attractive prospects for the difficult problem of preventing and controlling corn soil-borne diseases. Summary of the invention

[0005] In view of this, the object of the present invention is to provide a strain of Bacillus Velez ZJUHUCC1 and its application; the Bacillus Velez ZJUHUCC1 can significantly promote the growth of corn crops, improve the resistance hormone and systemic resistance levels of corn, and reduce the ability of southern root-knot nematodes to infect corn.

[0006] The invention provides a strain of Bacillus velezensis ZJUHUCC1. The deposit number of the Bacillus velezensis ZJUHUCC1 is CCTCC NO: M 2025095.

[0007] The invention provides a microbial agent, comprising the Bacillus Velez subtilis ZJUHUCC1.

[0008] Preferably, the microbial agent is a liquid preparation.

[0009] Preferably, the live bacteria concentration of Bacillus Velezii ZJUHUCC1 in the microbial agent is 1×10 5 ~1×10 7 CFU / mL.

[0010] The present invention provides the use of the Bacillus Velezii ZJUHUCC1 or the microbial agent in promoting corn growth.

[0011] Preferably, the Bacillus Velez subtilis ZJUHUCC1 or the microbial agent can increase the auxin content in corn leaves and roots, and increase the chlorophyll content in leaves.

[0012] The present invention provides the use of the Bacillus Velez subtilis ZJUHUCC1 or the microbial agent in improving the level of corn resistance hormone.

[0013] Preferably, the corn resistance hormone comprises jasmonic acid-isoleucine conjugate and 12-oxophytodienoic acid.

[0014] The present invention provides the use of the Bacillus Velez subtilis ZJUHUCC1 or the microbial agent in improving the resistance of corn to southern root-knot nematodes.

[0015] The present invention provides a method for using the Velez Bacillus ZJUHUCC1 or the microbial agent, which comprises applying the Velez Bacillus ZJUHUCC1 or the microbial agent to the soil surface; applying the microbial agent 3 to 8 days after corn sowing; applying a volume of 8 to 12 ml / plant, and applying the microbial agent 1 to 2 times.

[0016] Compared with the prior art, the present invention has the following beneficial effects: the Velez subtilis ZJUHUCC1 provided by the present invention can promote corn growth, increase the content of auxin in corn leaves and roots, and increase the chlorophyll content of leaves; at the same time, it can increase the level of resistance hormones in corn leaves and roots, improve corn system resistance, and can also achieve the effect of inhibiting the infectivity of southern root-knot nematodes. The strain provided by the present invention is harmless to the environment and shows excellent potential in improving the growth of corn crops and preventing and controlling soil-borne nematode diseases.

[0017] At the same time, the present invention also prepares a microbial agent containing Bacillus velezensis ZJUCC1, which is harmless to the environment and has high safety, and will not cause environmental pollution. When applied to the soil surface, it can significantly promote the growth level of corn leaves and roots and the chlorophyll SPAD index after 48 hours, and the effect is fast; the growth-promoting effect is remarkable. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1 The dry weight of the aboveground part of corn plants at 48h, 72h, and 96h after application of Bacillus Velez-ZJUHUCC1;

[0019] Figure 2 It is the dry weight of underground part of corn plants at 48h, 72h, and 96h after application of Bacillus Velez-ZJUHUCC1;

[0020] Figure 3 It is the SPAD index of chlorophyll in the leaves of corn plants 48h, 72h, and 96h after application of Bacillus Velez ZJUHUCC1;

[0021] Figure 4 The number of root knots on maize roots after application of Bacillus Velez-ZJUHUCC1 (A) and the number of invasions of the second-instar larvae of southern root-knot insect (B);

[0022] Figure 5 The auxin content in the aerial parts of corn plants 48h and 72h after application of Bacillus Velez ZJUHUCC1;

[0023] Figure 6 The auxin content in the underground part of corn plants 48h and 72h after application of Bacillus Velez ZJUHUCC1;

[0024] Figure 7 The content of isoleucine-ribose conjugate in corn leaves 48h and 72h after application of Bacillus Velez-ZJUHUCC1;

[0025] Figure 8The content of 12-oxophytodienoic acid in the roots of corn plants 48h and 72h after application of Bacillus Velez-ZJUHUCC1.

[0026] Biological Deposit Description

[0027] Bacillus velezensis ZJUHUCC1 is deposited in the China Center for Type Culture Collection, the deposit address is Wuhan University, Wuhan, China, the deposit date is January 13, 2025, and the deposit number is CCTCC NO: M 2025095. DETAILED DESCRIPTION

[0028] The invention provides a strain of Bacillus velezensis ZJUHUCC1. The deposit number of the Bacillus velezensis ZJUHUCC1 is CCTCC NO: M 2025095.

[0029] The present invention also provides a microbial agent, comprising the Bacillus Velez subtilis ZJUHUCC1.

[0030] In the present invention, the microbial agent is preferably a liquid preparation; the concentration of live bacteria of Bacillus Velezii ZJUHUCC1 in the microbial agent is preferably 1×10 5 ~1×10 7 CFU / mL, more preferably (1 to 9)×10 6 The present invention has no special limitation on the culture method of the Bacillus Velezii ZJUHUCC1, and the conventional strain culture method in the art can be used.

[0031] The present invention provides the use of the Velez Bacillus ZJUHUCC1 or the microbial agent in promoting corn growth. In the present invention, the Velez Bacillus ZJUHUCC1 or the microbial agent can significantly increase the auxin content of corn leaves and roots; and increase the chlorophyll content of leaves.

[0032] The present invention provides the use of the Velez Bacillus ZJUHUCC1 or the microbial agent in improving the level of corn resistance hormone. In the present invention, the corn resistance hormone preferably includes jasmonic acid isoleucine conjugate and 12-oxophytodienoic acid (OPDA); further preferably, the Velez Bacillus ZJUHUCC1 can increase the content of jasmonic acid isoleucine conjugate in corn leaves and can also increase the content of 12-oxophytodienoic acid in corn roots.

[0033] The present invention provides the use of the Bacillus Velez subtilis ZJUHUCC1 or the microbial agent in improving the resistance of corn to southern root-knot nematodes.

[0034] The present invention provides a method for using the Velez Bacillus ZJUHUCC1 or the microbial agent, which comprises applying the Velez Bacillus ZJUHUCC1 or the microbial agent to the soil surface; applying the microbial agent 3 to 8 days after corn sowing; applying a volume of 8 to 12 ml / plant, and applying the microbial agent 1 to 2 times.

[0035] In the present invention, preferably, the Velez subtilis ZJUHUCC1 or the microbial agent is applied when the corn grows one leaf; the volume of the application is preferably 9-11 ml / plant, more preferably 10 ml / plant, and the number of applications is preferably 1-2 times. In the specific implementation of the present invention, the corn is planted in a pot, and the volume of the pot is preferably 350-450 ml, more preferably 380-420 ml, and more preferably 400 ml.

[0036] The technical solutions provided by the present invention are described in detail below in conjunction with the embodiments, but they should not be construed as limiting the protection scope of the present invention.

[0037] The microbial agent including the Bacillus Velez subtilis ZJUHUCC1 is hereinafter referred to as agent ZJUHUCC1.

[0038] Example 1

[0039] Preparation of bacterial agent ZJUHUCC1

[0040] Bacillus Velezii ZJUHUCC1 was inoculated into LB solid medium, activated at 30°C for 24 h, and a single colony was picked and inoculated into LB liquid medium, cultured at 30°C and 180 r / min for 24 h, centrifuged at 5000 r / min for 5 min, the cells were collected, washed twice with sterile water, and then sterile water was added to adjust the OD of the bacterial solution. 600 The value is 1.0, and a bacterial suspension is obtained.

[0041] Example 2

[0042] Corn planting and application of fungicide ZJUHUCC1

[0043] The corn seeds were soaked in sterile water for 12 hours and then transferred to a culture dish containing moist filter paper in a dark environment at 25°C for 24 hours. After germination, the corn seeds were sown in a black plastic pot with a volume of 400 ml filled with soil. The corn pot was placed in an artificial climate room at 25°C with a light intensity of 14 h / d. After 5 days, when the corn grew one leaf, 10 mL OD 600 The bacterial agent of 1.0 was added once (experimental group Ba), and the control group (CK) was added with an equal volume of sterile water. Each group was set up with 12 replicates.

[0044] Example 3

[0045] Experiment on promoting the growth of corn plants by using the fungal agent ZJUHUCC1

[0046] Corn was planted according to the method in Example 2 and the bacterial agent ZJUHUCC1 was applied on the soil surface. At 48h, 72h and 96h after the inoculation of Bacillus Velez ZJUHUCC1, the chlorophyll SPAD value data of the upper, middle and lower parts of the longest leaf of the corn were recorded with a chlorophyll meter. Then the soil in the black plastic flowerpot was poured out to obtain the plant, and the rhizosphere soil to which the root system adhered was rinsed with water. The aboveground and underground parts of the plant were separated, placed in a 60°C electric blast drying oven for drying for 3 days, and then the dry weight was measured and the data was recorded. The aboveground dry weight results of the corn plant are as follows: Figure 1 The dry weight of underground parts of maize plants is shown in Figure 2 The SPAD values ​​in corn leaves are shown in Figure 3 shown.

[0047] Table 1 Dry weight of aboveground part of corn plant (unit: g)

[0048]

[0049]

[0050] Table 2 Dry weight of underground part of corn plant (unit: g)

[0051]

[0052] Table 3 SPAD values ​​in corn leaves

[0053]

[0054]

[0055] The results showed that compared with the control group (CK), the dry weight of the aboveground and underground parts of corn in the treatment group (Ba) increased significantly at 48h, 72h, and 96h after inoculation with the ZJUHUCC1 inoculant, and the chlorophyll SPAD value in the leaves increased significantly, indicating that the inoculant ZJUHUCC1 can significantly promote the growth of corn.

[0056] Example 4

[0057] Experiment on the effect of fungal agent ZJUHUCC1 on the prevention and control of southern root-knot nematode infection

[0058] Corn was planted according to the method in Example 2 and the microbial agent was applied on the soil surface. The corn was illuminated for 14 h / d in an artificial climate chamber at 25°C. After 10 days of growth (i.e., after the third cotyledon was fully expanded), 5 positions were selected at the root of the planted corn, the sand was loosened downward by 2 cm, and 1000 second-instar larvae were inoculated in each pot. The sand was then filled to the root of the corn, and the control group was also inoculated with nematodes. After 10 days of inoculation with nematodes, the samples were collected, the roots were rinsed with running tap water until there was no sand on the roots, and the root surface water was wiped dry. The root knot number was counted, and the root knot number was used as a reproduction index of the nematode. The roots were placed in a -20°C refrigerator for 24 hours. The thawed root samples were cut into pieces with a particle size of 1 cm and placed in a blender. Tap water was added according to the ratio of 10 mL of tap water to 1 g of corn root. After sufficient stirring, the samples were passed through a nested mesh sieve with an upper mesh of 100 mesh and a lower mesh of 500 mesh. The residue on the 500-mesh mesh sieve was washed into a 50 mL centrifuge tube and counted under a microscope (OLYMPUS CKX53-HOUN) (the number of invading nematodes). The number of invasions was used as an infection indicator of southern root-knot nematodes. The results are shown in Figure 2. Figure 4 shown.

[0059] Table 4 Number of root knots and number of infection hatching in different treatment groups

[0060]

[0061] The results showed that compared with the control group, the number of root knots on the corn root system inoculated with the ZJUHUCC1 inoculant decreased by 53.75% and the invasion rate decreased by 45.21% after 10 days. Therefore, the strain ZJUHUCC1 can significantly inhibit the infestation of southern root-knot nematodes on corn.

[0062] Example 5

[0063] Plant hormone determination experiment

[0064] Plant corn according to the method in Example 2 and apply the bacterial agent on the soil surface. Collect the leaves and roots of corn 48h and 72h after inoculation, place them in liquid nitrogen for rapid freezing for 30min, grind them to obtain freeze-dried powder of corn root and leaf tissue. Prepare an internal standard solution in advance. The internal standard solution uses 100% methanol as solvent. The internal standard solution finally contains deuterated jasmonic acid-D5 (d5-JA), carbon 13 labeled jasmonic acid-isoleucine ( 13 C6-JA-Ile), deuterated salicylic acid-D4 (d4-SA), deuterated abscisic acid-D6 (d6-ABA) and deuterated indoleacetic acid-D5 (d5-IAA) were each 100 ng / mL. Then, a mixed standard was prepared, and each 1 mL of the mixed standard contained 990 μL of ethyl acetate as an extract and 10 μL of internal standard solution. Weigh 0.1g of corn leaf and root powder, add 1mL of mixed standard to every 0.1g of corn leaf and root powder, vortex on a vortexer 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 sampling bottle (2mL) with an inner tube (250μL) placed, so as to extract the auxin and resistance hormone content in the corn leaf and root powder, and analyze it by UHPLC-MS / MS to obtain the hormone content of the aboveground leaf part and underground root part of the corn after the application of the inoculant. The results of auxin content in corn leaves are as follows Figure 5 The results of auxin content in maize roots are shown in Figure 6 The results of jasmonic acid-isoleucine conjugate in corn leaves are shown in Figure 7 As shown, 12-oxophytodienoic acid in roots is Figure 8 shown.

[0065] The results showed that compared with the control group (CK), the contents of jasmonic acid isoleucine conjugates in the leaves and 12-oxophytodienoic acid in the roots of the treated group (Ba) increased significantly at 48h and 72h after inoculation with the ZJUHUCC1 bacterial agent, indicating that the strain ZJUHUCC1 can significantly improve the systemic basic resistance level of the aboveground and underground parts of corn.

[0066] It can be seen from the above embodiments that the Bacillus Velez ZJUHUCC1 provided by the present invention can promote corn growth, increase the content of auxin in corn leaves and roots, while increasing the level of resistance hormones in corn leaves and roots, improving corn systemic resistance, and can also achieve the effect of inhibiting the infection ability of southern root-knot nematodes.

[0067] 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 principle of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. A strain of Bacillus velezensis ZJUHUCC1, characterized in that: The deposit number of the Bacillus velez ZJUHUCC1 is CCTCC NO: M 2025095.

2. A microbial agent, characterized in that: It comprises the Bacillus Velez subtilis ZJUHUCC1 described in claim 1.

3. The microbial agent according to claim 2, characterized in that: The microbial agent is a liquid preparation.

4. The microbial agent according to claim 3, characterized in that: The live bacteria concentration of Bacillus Velez ZJUHUCC1 in the microbial agent is 1×10 5 ~1×10 7 CFU / mL.

5. Use of the Bacillus Velez subtilis ZJUHUCC1 described in claim 1 or the microbial agent described in any one of claims 2 to 4 in promoting corn growth.

6. The use according to claim 5, characterized in that: The Velez Bacillus ZJUHUCC1 or the microbial agent can increase the auxin content of corn leaves and roots, and increase the chlorophyll content of leaves.

7. Use of the Bacillus Velez subtilis ZJUHUCC1 described in claim 1 or the microbial agent described in any one of claims 2 to 4 in improving the level of corn resistance hormone.

8. The use according to claim 7, characterized in that: The corn resistance hormone includes jasmonic acid isoleucine conjugate and 12-oxophytodienoic acid.

9. Use of the Bacillus Velezii ZJUHUCC1 according to claim 1 or the microbial agent according to any one of claims 2 to 4 in improving the resistance of corn to southern root-knot nematodes.

10. A method for using the Bacillus Velezii ZJUHUCC1 according to claim 1 or the microbial agent according to any one of claims 2 to 4, characterized in that: The Velez Bacillus ZJUHUCC1 or the microbial agent is applied to the soil surface; the application time is 3 to 8 days after corn is sown; the application volume is 8 to 12 ml / plant, and the application frequency is 1 to 2 times.

Images