Westersic monocytogenes for preventing and treating root-knot nematode and application thereof

Abstract

The application discloses a huaxi brevitoxa for preventing and treating root-knot nematodes and application thereof. The huaxi brevitoxa (preserved in the Guangdong Microbial Digital Culture Collection Center, numbered as GDMCC No: 65867, and the preservation address is No. 100, Martyr Road, Yuexiu District, Guangzhou City, Guangdong Province) can not only attract nematodes but also kill nematodes, can actively attract nematodes to the pesticide action area, concentrate the nematodes for killing, improve the prevention and treatment effect, and can avoid the waste of traditional pesticide spraying in a large range, reduce the pesticide usage, reduce the cost, and reduce the environmental pollution. The lethal rate of the huaxi brevitoxa fermentation liquor to the root-knot nematodes is more than 89%, the lethal rate of the 50-fold diluted fermentation liquor to the root-knot nematodes is more than 80%, the action time is not more than 12 hours, the nematodes can be quickly killed before the larvae enter the plant, and the huaxi brevitoxa fermentation liquor has a good application prospect in the prevention and treatment of plant root-knot nematodes.

Description

TECHNICAL FIELD

[0001] The present application belongs to the field of microbial technology, and particularly relates to a west short-pore bacterium for preventing and treating root-knot nematode and application thereof. BACKGROUND

[0002] Plant root-knot nematode is a kind of fixed obligate endoparasitic nematode, which obtains nutrition from the host through feeding sites during parasitism, and the swollen feeding sites cause root swelling to form root knots, thereby damaging the plant's ability to absorb nutrients and water, and the damage symptoms include weak plant growth, wilting and yellowing of leaves, root deformation, low yield, poor fruit quality and other problems. The host range of root-knot nematode is very wide, including various vegetables such as cucumber, tomato, pepper and bean, as well as economic crops such as corn, potato, soybean and oilseed. In most areas of southern China and some warm areas in northern China, it is distributed almost everywhere on the earth except Antarctica. There is no doubt that parasitic nematodes have caused a lot of losses to many crops in temperate, subtropical and tropical regions. The total annual economic loss caused by 37 important crops worldwide is 358.24 billion US dollars, the annual loss of 20 basic food crops is estimated at 215.77 billion US dollars, and the annual loss of 17 important economic crops is estimated at 142.47 billion US dollars. In addition, the proportion of global important crop planting areas infected by plant nematodes is also at a high level, for example, 90% of the global banana planting areas have plant nematode diseases.

[0003] Root-knot nematode often occurs mixed with other diseases during crop planting, and plays an important role as a pioneer in opening up the way during this process. Root-knot nematodes cause a large number of wounds in the roots, allowing pathogens to invade the host using the wounds caused by nematode infection. The complex infection of multiple pathogens on the host shows various symptoms and forms a 1+1>2 consequence, which greatly aggravates the damage. For example, the pathogen Ralstonia solanacearum interacts with southern root-knot nematode. Ralstoma solanacearum With global warming, the increase of protected area, and perennial monoculture, combined with the complexity of soil environment, root complex disease has become the norm, which seriously restricts China's agricultural production.

[0004] *Bacillus cereus*, a Gram-negative bacterium belonging to the genus *Bacillus* and family *Stenobacteria*, was first proposed by Segers et al. The genus *Bacillus* comprises 33 species and can be found in various environments, such as soil, water, activated sludge, and plant roots. *Bacillus cereus* has broad application potential, including cadmium biosorption, soil bioremediation, water pollution treatment, and promoting plant growth in sustainable agriculture in arid regions. For humans, *Bacillus cereus* is an opportunistic pathogen that can cause a range of hospital-acquired infections, such as bacteremia, eye infections, peritonitis, urinary tract infections, and skin and soft tissue infections. However, reports on the control of nematodes by *Bacillus cereus* are limited.

[0005] The present invention aims to provide a new strain of *Shortwave Monoclonalella spp.* with excellent nematode-attracting and killing effects. Summary of the Invention

[0006] The first objective of this invention is to provide a *Shortwave Monoclonalella* strain (…). Brevundimonas huaxiensis )1.547-1, the second object of the present invention is to provide the aforementioned *Shortwave Monoclonalella spp.* ( Brevundimonas huaxiensis Application of 1.547-1.

[0007] The first objective of this invention is achieved as follows: a strain of *Syntrophus spp.* (West China shortwave monoclonal antibody) Brevundimonas huaxiensis 1.547-1 ， It is deposited at the Guangdong Provincial Center for Microbial Culture Collection, with the number GDMCC No: 65867, on January 16, 2025, at No. 100 Xianlie Middle Road, Yuexiu District, Guangzhou City, Guangdong Province.

[0008] The second objective of this invention is achieved by applying the *Syntrophus huaxiensis* 1.547-1 in the control of plant root-knot nematodes.

[0009] The beneficial effects of this invention are as follows:

[0010] 1. The *Shortwave Monoclonalella* strain provided by this invention (… Brevundimonas huaxiensis Strain 1.547-1 was derived from the nematode fungus Oligosporium argenteum (…). Arthrobotrys oligospora 1.5470 is an endophytic bacterium of this fungal strain. The two form a symbiotic relationship, which allows this strain to assist the nematode-eating fungus in inhibiting the presence of nematodes to a certain extent. This improves the environmental control ability of the nematode-eating fungus Oligosporium nobile or this strain as a biocontrol agent, thereby improving the control efficiency of the nematode-eating fungus strain and this strain as a biocontrol agent against nematodes.

[0011] 2. The fermentation broth of the Brevundimonas haworthensis provided by the application can not only attract nematodes but also kill nematodes, can actively attract nematodes to the drug action area, concentrate on killing, improve the control effect, and can avoid the waste of traditional pesticide spraying in a large range, reduce the amount of pesticide used, reduce the cost, and reduce environmental pollution.

[0012] 3. The fermentation broth of the Brevundimonas haworthensis provided by the application has a mortality rate of more than 89% on root-knot nematodes, a mortality rate of more than 80% on root-knot nematodes when the fermentation broth is diluted 50 times, and an action time of no more than 12 hours, can quickly kill nematodes before the larvae enter the plant, and has a good application prospect in the control of plant root-knot nematodes. BRIEF DESCRIPTION OF DRAWINGS

[0013] Figure 1 Fig. A is a micrograph of the endoparasitic Brevundimonas haworthensis 1.547-1, and Figs. C and D are fluorescence morphological diagrams of the Brevundimonas haworthensis 1.547-1;

[0014] Figure 2 Fig. 2 is a phylogenetic tree of the Brevundimonas haworthensis 1.547-1;

[0015] Figure 3 Fig. 3 is a picture of the Brevundimonas haworthensis 1.547-1 culture solution attracting nematodes, wherein Fig. a is a two-part plate, and Fig. b is an enlarged picture of nematodes crossing the plate midline and crawling to the strain growth area; the left side of the two-part plate is the strain growth area, and the right side is the nematode living area. DETAILED DESCRIPTION

[0016] The application will be further described in detail below in combination with the drawings and examples, but the application is not limited in any way by the description, and any transformation or improvement based on the teaching of the application falls within the protection scope of the application.

[0017] The application provides a Brevundimonas haworthensis (Brevundimonas haworthensis) 1.547-1 Brevundimonas huaxiensis ) 1.547-1 ， The strain is preserved in the Guangdong Microbial Culture Collection Center, and the number is GDMCC No: 65867, and the preservation address is No. 100, Martyrs Road, Yuexiu District, Guangzhou City, Guangdong Province.

[0018] The application also provides application of the Brevundimonas haworthensis 1.547-1 in the control of plant root-knot nematodes.

[0019] The application is to kill root-knot nematodes by metabolites produced by culture of the Brevundimonas haworthensis 1.547-1.

[0020] The application is to control plant root-knot nematodes by metabolites produced by culture of the Brevundimonas haworthensis 1.547-1.

[0021] The application is to make the preparation of Brevundimonas vesiculosa 1.547-1 or the preparation of Brevundimonas vesiculosa 1.547-1 combined with the nematophagous fungus Arthrographis glomerata to prevent and control plant root-knot nematodes.

[0022] The application further provides a bacterial agent prepared based on the Brevundimonas vesiculosa 1.547-1.

[0023] The preparation method of the bacterial agent is as follows:

[0024] 1) The Brevundimonas vesiculosa 1.547-1 strain is transferred to a seed culture medium, and then cultured at a speed of 200 r / min on a shaker at 36-38 DEG C for 20 h to obtain a fermentation seed liquid;

[0025] 2) The fermentation seed liquid is inoculated into a fermentation culture medium, and then cultured at a speed of 185 r / min on a shaker at 37 DEG C for 40 h to obtain a fermentation liquid, which is the target bacterial agent.

[0026] In step 1), the seed culture medium is an LB culture medium.

[0027] In step 2), the fermentation culture medium is an LB culture medium.

[0028] Example 1: Strain separation, purification and identification

[0029] The Brevundimonas vesiculosa strain of the application is separated from the nematophagous fungus Arthrographis glomerata (Yun et al., 2014) collected in Xishuangbanna, Yunnan Province, China. Arthrobotrys oligospora The fungus strain is collected in the "Southwest Germplasm Resource-Microorganism Bank" (the bank is managed and operated by the State Key Laboratory for Conservation and Utilization of Biological Resources of Yunnan University), and the original number of the strain is 1.5470, and the preservation number is YMF1.05470.

[0030] Strain separation method: the fungus block is inoculated into a CMB liquid culture medium, and then cultured on a constant-temperature shaker (28 DEG C, 180 rpm / min) for 10 d, and then the mycelium in the shake flask is extracted by a vacuum filtration pump and transferred into a 2 mL sterile centrifuge tube, and sterile deionized water and sterile steel balls are added, and the collected mycelium is crushed by using a high-throughput tissue crusher to obtain a fungus tissue homogenate, and then a proper amount of the fungus tissue homogenate is added dropwise on LB, TWYE and YEM solid culture media, and then the culture media are evenly coated by using a coater, and then the plates are sealed by using Parafilm, and then the plates are cultured in a constant-temperature incubator at 30 DEG C for 3-4 d, and then the growth of the colonies is observed and recorded, and then a single colony is picked on LB solid culture medium according to the morphological characteristics of the bacteria, and the operation is repeated to obtain the Brevundimonas vesiculosa 1.547-1. According to the molecular biology identification, the identification result shows that the strain 1.547-1 is a new species of Brevundimonas vesiculosa, and the ITS sequence of the strain 1.547-1 is shown as SEQ ID No. 1.

[0031] The medium components used for strain isolation and culture are as follows:

[0032] 1. Fungal medium:

[0033] CMB liquid medium: corn meal 20 g, add water to 1 L. Autoclave at 121 °C for 20 min.

[0034] 2. Bacterial medium: LB, TWYE and YEM

[0035] LB medium: yeast extract 5 g, tryptone 10 g, sodium chloride 10 g, add water to 1 L. Autoclave at 121 °C for 20 min.

[0036] TWYE medium: yeast extract 0.25 g, potassium phosphate dibasic 0.5 g, add water to 1 L. Autoclave at 121 °C for 20 min.

[0037] YEM medium: yeast extract 0.5 g, mannitol 5 g, potassium phosphate dibasic 0.5 g, magnesium sulfate heptahydrate 0.2 g, sodium chloride 0.1 g, adjust pH to 7.0, add water to 1 L. Autoclave at 121 °C for 20 min.

[0038] NGM solid medium: tryptone 2.5 g, sodium chloride 3 g, agar 15 g, add deionized water to 1 L. After sterilization at 121 °C for 20 min, when the temperature drops to about 55 °C, add 1 mL each of 1M phosphate buffer, 1M magnesium sulfate solution, 1M calcium chloride solution and 1 mL of 5 mg / mL cholesterol (dissolved in anhydrous ethanol) solution treated by 0.22 μm microporous filter, shake evenly and pour flat.

[0039] Example 2 Fermentation broth preparation

[0040] 1. Seed culture: transfer pure strain 1.547-1 on the plate of Example 1 to multiple 250 mL flasks containing 100 mL of seed medium (LB medium), then culture at 37 °C ± 1 °C under reciprocating shaker at 200 r / min for 20 h to obtain the fermentation seed broth.

[0041] 2. Fermentation culture: inoculate the prepared fermentation seed broth into fermentation medium (LB medium) in a 250 mL flask

[0042] with a liquid volume of 150 mL and an inoculation amount of 5%, culture at 37 °C and a rotation speed of 185 r / min for 40 h to obtain the fermentation broth.

[0043] Experimental Example 1

[0044] Nematode attraction test on the fermentation broth prepared in Example 2

[0045] Test method: Sterilized and melted NGM medium was injected in advance into a two- compartment petri dish with a diameter of 9 cm, and used after solidification. 100-150 2nd instar southern root-knot nematodes were added at the center point of one compartment; 20 μL of bacterial solution (OD 600 = 1.0) was added at the center point of the other compartment and evenly coated with a coating rod, and after the liquid stopped flowing, all test plates were immediately sealed with Parafilm and placed in a 22°C incubator for culture, and the number of nematodes crossing the compartment to the bacterial solution end was observed under a stereomicroscope at 2 h, 4 h, and 8 h, respectively, the attraction index (AR) was calculated and recorded, and E. coli OP50 and blank culture solution LB were used as controls. The test was repeated 5 times.

[0046]

[0047] The larger the AR value, the stronger the attraction of the endophytic bacteria to the nematodes.

[0048] Table 1 Results of the attraction test of the fermentation broth prepared in Example 2 on nematodes

[0049]

[0050] From Figure 3 It can be seen that the nematodes move to the left area and even crawl over the middle partition under the attraction of the fermentation broth.

[0051] The results in Table 1 show that the AR value of the fermentation broth prepared in Example 2 is >0.3 at 4 h and >0.5 at 8 h, and the fermentation broth has a significant attraction to nematodes. The AR value of the control is zero, indicating that the control has no attraction.

[0052] Example 2 Lethality test of the fermentation broth prepared in Example 2 on nematodes

[0053] Test method: 700 μL of fermentation broth was added to a cell culture dish, and then a root-knot nematode suspension (20 μL: 80-100 2nd instar southern root-knot nematodes) was added, mixed well, and the test cell culture dish containing the bacterial culture solution and nematodes was placed in a 22°C incubator for culture, and the number of live and dead nematodes was recorded under a stereomicroscope every 30 minutes. If the nematodes were found to be rigid, and did not respond to stimulation with a needle, it was considered that the nematodes had died. Blank culture solution and E. coli OP50 were used as controls, and the mortality rate and corrected mortality rate of nematodes were calculated according to Zhang et al. (2016). The test was repeated 5 times. The results are shown in Table 2.

[0054]

[0055]

[0056] Table 2 Results of lethal test of the fermentation broth prepared in Example 2 on plant root-knot nematodes

[0057]

[0058] As shown in Table 2, the 12h lethal rate of the fermentation broth prepared in Example 2 on southern root-knot nematodes was over 89%.

[0059] The fermentation broth prepared in Example 2 was diluted 50 times and then subjected to the nematode lethal test according to the above method, and the test results were as follows:

[0060] As shown in Table 3.

[0061] Table 3 Results of lethal test of the diluted broth (x50) of the fermentation broth prepared in Example 2 on plant root-knot nematodes

[0062]

[0063] As shown in Table 3, the 12h lethal rate of the fermentation broth prepared in Example 2 diluted 50 times on the 2nd instar larvae of southern root-knot nematodes was over 80%.

[0064] Experimental Example 3 Field Test

[0065] 1. Test microbial agent: fermentation broth prepared in Example 2

[0066] Control group: 2% abamectin suspension (Jiangsu Shuangning Chemical Industry)

[0067] 2. Test treatment

[0068] Treatment 1: fermentation broth prepared in Example 2 10ml / plant

[0069] Treatment 2: fermentation broth prepared in Example 2 20ml / plant

[0070] Treatment 3: fermentation broth prepared in Example 2 30ml / plant

[0071] Treatment 4: fermentation broth prepared in Example 2 40ml / plant

[0072] Treatment 5: fermentation broth prepared in Example 2 50ml / plant

[0073] Positive control: 2% abamectin suspension 20ml / plant

[0074] Blank control: 50ml water / plant

[0075] 3. Experimental method

[0076] The test crop was tomato, and the infection was southern root-knot nematode (Meloidogyne incognita) Meloidogyne incongnita), yellow loam. The test plot area is 100 square meters, 50 seedlings per treatment, seedling distance 50 cm, plot arrangement adopts random arrangement method. At the time of tomato seedling transplanting, the pesticide is applied to the rhizosphere, and the experimental period is managed according to the routine.

[0077] 4. Investigation method

[0078] After the tomato is completely harvested, the incidence, disease index, and relative control effect are investigated.

[0079]

[0080]

[0081]

[0082]

[0083] Table 4 Disease grading standard

[0084]

[0085] 5. Experimental results

[0086] Table 5 Field use of the fermentation liquid prepared in Example 1 for the control effect of root-knot nematode

[0087]

[0088] The results in Table 5 show that, compared with the control, the relative control effect of the fermentation liquid prepared in Example 2 on the field nematode at five concentrations is better than that of the same period of abamectin (mainly used for field nematode control).

Claims

1. A strain of *Synthetium spp.* that can attract and kill plant root-knot nematodes ( Brevundimonas huaxiensis Strain 1.547-1 is deposited at the Guangdong Provincial Microbial Culture Collection Center, with the number GDMCC No: 65867, located at No. 100, Xianlie Middle Road, Yuexiu District, Guangzhou City, Guangdong Province.

2. A *Shortwave Monoclonalella* strain as described in claim 1 (… Brevundimonas huaxiensis Application of strain 1.547-1 in the control of plant root-knot nematodes.

3. The application according to claim 1, characterized in that, With the aforementioned *Shortwave Monoclonalella* ( Brevundimonas huaxiensis A microbial agent for controlling plant root-knot nematodes was prepared using strain 1.547-1. The preparation method of the microbial agent is as follows: 1) *Syntrophus spp.* (West China shortwave monocytogenes) Brevundimonas huaxiensis The strain 1.547-1 was transferred into the seed culture medium and then cultured on a shaker at 35-37℃ at a speed of 150-200 r / min for 20 h to obtain the fermentation seed liquid; 2) Inoculate the fermentation seed liquid into the fermentation medium and culture it on a shaker at 35~37℃ at a speed of 150~200r / min for 40~72 hours. The resulting fermentation liquid is the target inoculum.

4. The application according to claim 3, characterized in that, In step 1), the seed culture medium is LB medium.

5. The application according to claim 3, characterized in that, In step 2), the fermentation medium is LB medium.

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