A strain of Priesteria afroides HN02 and its application in controlling plant root-knot nematode disease

Abstract

The present invention provides a strain of Priestia aryabhattai HN02 and an application thereof, which is named as Priestia aryabhattai HN02 and has a deposit number of CGMCC NO. 28379. The strain has a good toxic effect on second-instar larvae and eggs of root-knot nematodes, is a biocontrol strain with high toxicity to plant parasitic nematodes, not only enriches the biocontrol bacteria resources of plant parasitic nematodes, but more importantly, has a very important significance for the development of green pesticides or fertilizers for killing nematodes.

Description

Technical Field

[0001] The invention relates to the technical field of microbiology, and in particular to a strain of Priesteria afroides HN02 and application thereof in preventing and controlling plant root-knot nematode disease. Background Art

[0002] At present, more than 80 species of root-knot nematodes have been reported, mainly parasitizing more than 2,000 hosts such as food crops and cash crops. Among them, the southern root-knot nematode (Meloidogyne incognita) is the most widely distributed and most harmful plant parasitic nematode, mainly distributed in tropical, subtropical and temperate regions. Root-knot nematodes attack the root system of plants, absorb a large amount of nutrients to maintain their own development, cause plant photosynthesis disorders, and reduce the plant's ability to absorb water and nutrients to harm host plants. At the same time, the defense system of crops invaded by nematodes is damaged, making them more vulnerable to attacks by other pathogens in the soil, resulting in root and stem rot, poor growth, root rot, dead branches and leaves, yellowing, twisting and wilting, short plants, slow growth and other phenomena, which seriously affect crop growth and yield. In general years, the yield reduction is 10% to 15%, and in severe cases it can reach 30% to 40%, or even a total crop failure. The economic losses caused each year are as high as 100 billion US dollars. It is one of the major plant health problems in the world. The prevention and control of root-knot nematode disease is imminent.

[0003] The application of chemical agents is currently the main measure for the prevention and control of root-knot nematodes. Although its effect is quite significant, long-term and large-scale use can easily cause root-knot nematodes to develop resistance, while also destroying the ecology and endangering the health of humans and livestock. In recent years, seeking biological control to replace chemical control has become a major trend at home and abroad. With the requirements of the sustainable development strategy and the improvement of the quality of public life, research has found that biocontrol bacteria are safe and friendly to the ecological environment and are considered to be an important alternative to chemical control of root-knot nematode disease. In addition, many biocontrol bacteria have the function of promoting plant growth while inhibiting the occurrence of the disease. Therefore, how to screen and identify biocontrol bacteria from soil or plant materials can provide new basis and ideas for the development of biocontrol bacteria and green control of root-knot nematode disease.

[0004] Therefore, it is necessary to screen and identify a strain that can prevent and control root-knot nematodes from soil or plant materials, so as to provide new basis and ideas for the development of biocontrol bacteria and green control of root-knot nematode disease. Summary of the invention

[0005] The purpose of the present invention is to provide a strain of Priesteria afroyi HN02 and its application. The strain of Priesteria afroyi HN02 has good control and killing effects on root-knot nematodes and can be used in biological control of root-knot nematodes in various plants.

[0006] In order to achieve the above object, the present invention adopts the following technical solution:

[0007] In a first aspect of the present invention, a strain of Priestia aryabhattai HN02 is provided. The classification name of the Priestia aryabhattai HN02 is Priestia aryabhattai, and the deposit number is CGMCC NO.28379.

[0008] In a second aspect of the present invention, a fermentation agent is provided, the fermentation agent comprising:

[0009] Fermenting and culturing the Affymetrium HN02 to obtain a fermentation liquid or a bacterial suspension;

[0010] Or the fermentation liquid is spray-dried to obtain a dry powder bacterial agent.

[0011] Furthermore, the preparation method of the bacterial suspension comprises:

[0012] The apricot bacterium HN02 is inoculated into a liquid culture medium for fermentation to obtain a fermentation liquid.

[0013] Furthermore, the fermentation culture conditions include: temperature of 30-35°C and pH of 5-9.

[0014] Furthermore, the formula of the liquid culture medium is: 4wt% to 12wt% glucose, 4wt% to 10wt% beef extract, 2wt% to 5wt% calcium carbonate, 2wt% to 5wt% potassium chloride, 1000mL deionized water, adjusted with hydrochloric acid to an initial pH of 7.0 to 7.2, and sterilized at 120°C for 20 to 25 minutes.

[0015] Furthermore, the preparation method of the dry powder bacterial agent includes:

[0016] 300 mL to 600 mL of the fermentation liquid is added to 350 g to 550 g of the carrier material, stirred at 35 to 40° C. and 80 to 200 r / min until the carrier material becomes flocculent, dried at 30 to 40° C., pulverized by air flow, and sieved to obtain a dry powdered bacterial agent of Priesteria aegypti HN02;

[0017] The preparation method of the carrier material comprises: mixing the carrier, the wetting agent, the dispersant and the protective agent in a mass ratio of 50% to 70%: 10% to 25%: 1% to 4%: 1% to 4%, spray drying and sieving to obtain the carrier material, wherein the air inlet temperature in the spray drying is 170 to 190° C., and the air outlet temperature is 55 to 65° C.

[0018] Further, the carrier includes at least one of white carbon black, diatomaceous earth, kaolin and bentonite;

[0019] The wetting agent includes at least one of fatty alcohol polyoxyethylene ether (AEO), sucrose fatty acid ester, Tween 80, ethoxylated alkyl sodium sulfate, sodium dodecylbenzene sulfonate, and sodium lignin sulfonate;

[0020] The dispersant includes at least one of calcium chloride, polyvinyl alcohol, sodium tripolyphosphate (STPP), soluble starch, ammonium sulfate, and polyethylene glycol;

[0021] The protective agent includes at least one of dextrin, silicon dioxide, hydroxymethyl cellulose, sodium hydroxymethyl cellulose and L-ascorbic acid.

[0022] In the third aspect of the present invention, provided is the use of the Prioccidioides aeruginosa HN02 or the fermentation agent in preventing and controlling plant root-knot nematode disease.

[0023] Furthermore, the plant root-knot nematode disease includes at least one of tobacco root-knot nematode disease, pepper root-knot nematode disease, cucumber root-knot nematode disease, and peanut root-knot nematode disease.

[0024] One or more technical solutions in the embodiments of the present invention have at least the following technical effects or advantages:

[0025] 1. The present invention provides a strain of Priestia aryabhattai HN02 and its application in preventing and controlling plant root-knot nematode disease. A strain of Priestia aryabhattai was isolated and screened from soil. The strain has good lethal activity against root-knot nematodes and eggs, and is a biocontrol strain with high activity against plant parasitic nematodes. It not only enriches the biocontrol bacteria resources for plant parasitic nematodes, but more importantly, is of great significance for the development of green pesticides or fertilizers for killing nematodes.

[0026] 2. The microbial biocontrol agent prepared by the present invention can provide nutrients and a colonization environment for the colonization of biocontrol bacteria, prolong the service life of the biocontrol bacteria, and has a small dosage and good safety for plants. It is environmentally friendly and pollution-free during use, has the advantages of being environmentally friendly, simple to apply, small dosage, and having a long lasting effect, and is easy to industrialize.

[0027] 3. The strain of Priesteria axyphylla HN02 provided by the present invention has a broad spectrum of nematode diseases. The laboratory has determined that it has good prevention and control effects on root-knot nematode diseases of many plants such as tobacco root-knot nematode disease, pepper root-knot nematode disease, peanut root-knot nematode disease, tomato root-knot nematode disease, etc., and the strain is stable.

[0028] The deposit date of Priestia aryabhattaii HN02 of the present invention is September 7, 2023, and the deposit number is CGMCCNO.28379. Its classification name is Priestia aryabhattaii, and the name of the depository is China General Microbiological Culture Collection Management Center, with an address of No. 1, Beichen West Road, Chaoyang District, Beijing, Postal Code: 100101. BRIEF DESCRIPTION OF THE DRAWINGS

[0029] In order to more clearly illustrate the technical solutions in the embodiments of the present invention, the following briefly introduces the drawings required for use in the description of the embodiments. Obviously, the drawings described below are some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without creative work.

[0030] Figure 1 A multi-gene combined phylogenetic tree of the strain HN02 of the present invention;

[0031] Figure 2 The present invention shows the potted control effect of the HN02 fermentation liquid of the strain on tobacco root-knot nematode disease. DETAILED DESCRIPTION

[0032] The present invention will be described in detail below in conjunction with specific implementations and examples, and the advantages and various effects of the present invention will be more clearly presented. It should be understood by those skilled in the art that these specific implementations and examples are used to illustrate the present invention, rather than to limit the present invention.

[0033] Throughout the specification, unless otherwise specifically stated, the terms used herein should be understood as meanings commonly used in the art. Therefore, unless otherwise defined, all technical and scientific terms used herein have the same meanings as those generally understood by those skilled in the art to which the present invention belongs. In the event of a conflict, the present specification takes precedence.

[0034] Unless otherwise specified, various raw materials, reagents, instruments and equipment used in the present invention can be purchased from the market or obtained by existing methods.

[0035] The following will describe in detail a strain of Priesteria afrozenii HN02 and its application in the present application in combination with examples and experimental data.

[0036] Example 1: Isolation, purification and identification of Priesteria afrozena HN02

[0037] 1. Isolation and purification of strain HN02

[0038] Weigh 10 g of soil sample from a plot in Hainan where tomato root-knot nematodes are more likely to infect, mix thoroughly with 90 mL of sterile water, and place in a 28°C constant temperature shaker for 2 h to obtain a soil suspension. Dilute with sterile water to 10 5 , 10 6 , 10 7 , 10 8 , use a coating rod to spread on a solid sterile NA plate. After culturing in a 30℃ incubator for 3 days, pick single colonies with different morphologies on the plate and place them on a solid sterile NA plate for streaking purification. Store the purified single colonies in a 4℃ refrigerator for strain screening.

[0039] 2. Screening of strain HN02

[0040] Take 500 μL of fermentation liquid of the isolated strain and add it to a 48-well plate, then add 50 μL of second-instar larvae suspension (100) to each well. Set liquid NA culture medium as the control and repeat 3 times. After culturing in a 25°C incubator for 24 hours, observe the activity of nematodes under a microscope, stimulate the nematodes with 1 mol / L NaOH, and stiffness is considered death. Calculate the nematode mortality rate and corrected mortality rate, mortality rate (%) = number of nematode deaths / total number of nematodes × 100, corrected mortality rate (%) = (mortality rate of nematodes in the treatment group - mortality rate of nematodes in the control group) / (1-mortality rate of nematodes in the control group) × 100. The results are shown in Table 1.

[0041] Table 1 Effects of different bacterial fermentation broths on the mortality of root-knot nematodes

[0042]

[0043] As shown in Table 1, a total of 8 bacterial strains were isolated from Hainan red soil where root-knot nematode disease was more serious. Among them, the No. 5 strain fermentation liquid had the highest corrected mortality rate for the second-instar larvae after 24 hours of treatment, which was 96.21%, significantly higher than the other strains. Therefore, the No. 5 strain was used as the test strain for the next study. Next, the No. 5 strain was named strain HN02 for the next experiment.

[0044] 2. Identification of strain HN02

[0045] (1) Morphological characteristics of strain HN02: The colonies of strain No. 5, named HN02, on NA solid plates were round and light yellow; Gram staining was positive, indicating that it was a Gram-positive bacterium.

[0046] (2) Physiological and biochemical characteristics of strain HN02: The growth temperature of strain HN02 is 25-37°C, the pH is 5-9, the optimal growth temperature is 30°C, and the optimal pH is 7.

[0047] (3) 16S rDNA sequence determination and analysis of strains: The genomic DNA of the strain was extracted using the Bacteria Genomic DNA Kit (Beijing Quanshijin Biotechnology Co., Ltd.), and 16S rDNA (universal primers 27F / 1492R) and rpoB and rpoD genes were amplified by PCR. The PCR reaction conditions were: 94°C for 5 min; 94°C for 1 min, 56°C for 1 min, 72°C for 90 s, 35 cycles; 72°C for 7 min. The PCR amplification products were sequenced by Qingdao Paisono Gene Technology Co., Ltd., and the sequences were submitted to GenBank for BLAST comparison and phylogenetic analysis by MEGA 6.0 software.

[0048] The sequencing result (as shown in SEQ ID NO.1) was compared with BLAST in NCBI, and it was found that the sequence homology between the strain and Priestia aryabhattai was 99.6%. Combined with the morphology and physiological and biochemical characteristics of the strain, the strain was determined to be Priestia aryabhattai. The strain has been deposited in the China General Microbiological Culture Collection Center with the deposit number CGMCC NO.28379.

[0049] Example 2: In vitro activity determination of HN02 strain fermentation broth at different concentrations against root-knot nematodes

[0050] The strain HN02 was inoculated into a liquid culture medium with the following formula: 8wt% glucose, 10wt% beef extract, 5wt% calcium carbonate, 2wt% potassium chloride, and 1000mL deionized water. The initial pH was adjusted to 7.0-7.2 with hydrochloric acid and sterilized at 120℃ for 20-25min. After culturing at 32℃ and 180r / min for 16-28h, the concentration of the bacterial solution was tested. When the content of the bacterial solution exceeded 1×10 8 cfu / mL, it is the fermentation broth of strain HN02, which is diluted into 5-fold and 10-fold dilutions with sterile water. 500 μL of the fermentation broth stock, 5-fold dilution and 10-fold dilution were respectively added to a 48-well plate, and 50 μL (100) of the second-instar larvae suspension was added to each well. NA medium was used as the control and repeated 3 times. After 24 and 48 hours, the death of the second-instar larvae was observed, and the nematode mortality rate and corrected mortality rate were calculated. The results are shown in Table 2.

[0051] Table 2 Effects of different concentrations of fermentation broth of strain HN02 on nematode mortality

[0052]

[0053] As shown in Table 2, the corrected mortality of root-knot nematodes with different concentrations of HNO2 fermentation broth decreased significantly with the increase of dilution multiple. After 24 hours of treatment with HNO2 fermentation filtrate original solution, 5-fold dilution and 10-fold dilution, the corrected mortality of second-instar larvae was 97.07%, 79.05% and 55.07%, respectively. After 48 hours of treatment, the corrected mortality of second-instar larvae was 100.00%, 81.89% and 61.01%, respectively.

[0054] Example 3: Preparation of Priesteria afroicola HN02 powder

[0055] 70% white carbon black, 20% sodium lignin sulfonate, 3% polyvinyl alcohol, 5% sucrose and 2% hydroxymethyl cellulose are mixed evenly and spray dried, with the air inlet temperature at 180°C and the air outlet temperature at 60°C. The obtained powder is sieved through a 400-mesh sieve to prepare a powder carrier. 300mL to 600mL of the fermentation liquid of the strain HN02 is added to 350g-550g of the carrier material (specifically 500mL:500g is selected), stirred at 32°C, 150r / min for 30min, placed in a 40°C electric constant temperature blast drying oven for drying, crushed by a jet mill, and sieved through a 400-mesh sieve to obtain HN02 powder.

[0056] Example 4: Application of Priesteria afroides HN02 in preventing and controlling plant root-knot nematode disease

[0057] After 4 weeks of tobacco seedling emergence, the tobacco seedlings were transplanted into plastic pots with a diameter of 10 cm and 300 g of soil. The pot experiment was set up with the following treatments:

[0058] HNO2 fermentation broth (1×10 8 cfu / mL), HN02 powder, 2% avermectin emulsifiable concentrate and water. One week after tobacco transplanting, roots were irrigated with 50 mL / plant of HN02 fermentation liquid, 50 mL / plant of HN02 powder diluted 500 times, and 50 mL / plant of 2% avermectin emulsifiable concentrate diluted 500 times, respectively. 50 mL / plant of clean water was used as the control. 10 times were repeated. After 2 days of root irrigation for each treatment, two 1 cm deep holes were symmetrically made at a distance of 2 cm from the roots of the tobacco plants, and 5 mL of nematode suspension was injected. Each plant was inoculated with 1000 nematodes. After 30 days, the plant height and fresh weight of tobacco plants were counted, and the root knot index and control effect were calculated. The root knot index = Σ (disease level value × number of diseased plants at the corresponding level) / (total number of plants investigated × highest disease level) × 100, and the control effect (%) = (root knot index of control group - root knot index of treatment group) / root knot index of control group × 100.

[0059] Table 3 Control effect of strain HN02 on tobacco root knot nematode disease

[0060]

[0061] As shown in Table 3, the root knot index of tobacco in the root irrigated treatment with the fermented liquid of strain HN02 and the diluted liquid of HN02 powder was 11.3 and 18.1, respectively, which was significantly lower than 80.1 of the control, and the control effect reached 85.9% and 77.4% in 30 days; while the root knot index of the root irrigated treatment with 2% avermectin diluted liquid was 35.2, and the control effect was 56.1%, which showed that strain HN02 had a good control effect on root-knot nematodes. The plant height and fresh weight of tobacco treated with the fermented liquid of strain HN02 and the diluted liquid of HN02 powder were significantly higher than those of the control group, both increased by more than 55%, indicating that strain HN02 had a good growth-promoting effect.

[0062] In summary, the present application isolates and screens Apricotella HN02 from the soil infected with root-knot nematodes in Hainan Province, and its fermentation liquid has a high lethal effect on the second-instar larvae of root-knot nematodes, with a 24-hour corrected mortality rate of 60.14% and a 48-hour corrected mortality rate of 83.39%. The fermentation liquid has a 30-day control effect on indoor potted tobacco root-knot nematodes, and the prepared HN02 powder reaches 77.4%. In the present application, Apricotella HN02 significantly increases the fresh weight and plant height of tobacco, promotes the growth and development of tobacco plants, and is of great significance for the development of green pesticides or fertilizers for killing nematodes.

[0063] The above embodiments are preferred implementation modes of the present invention, but the implementation modes of the present invention are not limited to the above embodiments. Any other changes, modifications, substitutions, combinations, and simplifications that do not deviate from the spirit and principles of the present invention should be equivalent replacement methods and are included in the protection scope of the present invention.

[0064] Finally, it should be noted that the terms "comprises," "includes," or any other variations thereof are intended to cover non-exclusive inclusion, such that a process, method, article, or apparatus that includes a series of elements includes not only those elements, but also other elements not explicitly listed, or also includes elements that are inherent to such process, method, article, or apparatus.

[0065] Although the embodiments of the present invention have been described, those skilled in the art may make additional changes and modifications to these embodiments once they have learned the basic creative concept. Therefore, the appended claims are intended to be interpreted as including the embodiments and all changes and modifications falling within the scope of the present invention.

[0066] Obviously, those skilled in the art can make various changes and modifications to the present invention without departing from the spirit and scope of the present invention. Thus, if these modifications and variations of the present invention fall within the scope of the claims of the present invention and their equivalents, the present invention is also intended to include these modifications and variations.

Claims

1. A strain of Priesteria afrozenii HN02, characterized in that: The Priestia aryabhattai HN02 is classified and named as Priestia aryabhattai, and its deposit number is: CGMCCNO.28379; The colonies of the Prioccidioides afroide HN02 on the NA solid plate were round and light yellow.

2. A fermentation agent, characterized in that: The fermentation agent comprises: A fermentation broth or bacterial suspension obtained by fermenting the Affymetrium HN02 according to claim 1; or spray-drying the fermentation liquid to obtain a dry powder bacterial agent; The preparation method of the bacterial suspension comprises: inoculating the Affymetrium HN02 described in claim 1 into a liquid culture medium for fermentation to obtain a fermentation liquid; The formula of the liquid culture medium is: 4wt% to 12wt% of glucose, 4wt% to 10wt% of beef extract, 2wt% to 5wt% of calcium carbonate, 2wt% to 5wt% of potassium chloride, 1000mL of deionized water, adjusted with hydrochloric acid to an initial pH of 7.0 to 7.2, sterilized at 120°C for 20 to 25min; The preparation method of the dry powder bacterial agent comprises: adding 300 mL to 600 mL of the fermentation liquid to 350 g to 550 g of a carrier material, stirring at 35 to 40° C. and 80 to 200 r / min until the carrier material becomes flocculent, drying at 30 to 40° C., air flow crushing, and sieving to obtain a dry powder bacterial agent of Priesteria aegypti HN02; The preparation method of the carrier material comprises: mixing the carrier, the wetting agent, the dispersant and the protective agent in a mass ratio of 50% to 70%: 10% to 25%: 1% to 4%: 1% to 4%, spray drying and sieving to obtain the carrier material, wherein the air inlet temperature in the spray drying is 170 to 190° C., and the air outlet temperature is 55 to 65° C.

3. The fermentation agent according to claim 2, characterized in that: The fermentation culture conditions include: temperature of 30-35°C and pH of 5-9.

4. The fermentation agent according to claim 2, characterized in that: The carrier includes at least one of white carbon black, diatomaceous earth, kaolin and bentonite; The wetting agent includes at least one of fatty alcohol polyoxyethylene ether, sucrose fatty acid ester, Tween 80, ethoxylated alkyl sodium sulfate, sodium dodecylbenzene sulfonate, and sodium lignin sulfonate; The dispersant includes at least one of calcium chloride, polyvinyl alcohol, sodium tripolyphosphate, soluble starch, ammonium sulfate, and polyethylene glycol; The protective agent includes at least one of dextrin, silicon dioxide, hydroxymethyl cellulose, sodium hydroxymethyl cellulose and L-ascorbic acid.

5. Use of the Afrococcus aegypti HN02 according to claim 1 or the fermentation agent according to any one of claims 2 to 4 in preventing and controlling plant root-knot nematode disease.

6. The use according to claim 5, characterized in that: The plant root-knot nematode disease includes at least one of tobacco root-knot nematode disease, vegetable root-knot nematode disease and peanut root-knot nematode disease.

Images