Nematicidal streptomyces JXSY860 and application thereof

Abstract

The invention discloses nematode-killing streptomyces JXSY860 and application thereof, and belongs to the technical field of microbial pesticides. According to the invention, a new strain of Streptomyces turiscabs JXSY860 is found, the new strain of Streptomyces turiscabs JXSY860 has been preserved in Guangdong Microbial Culture Collection Center on August 8, 2024, and the preservation number is GDMCC No: 64998. The invention also discloses a preparation method of the Streptomyces turiscabs JXSY860. Through verification, the JXSY860 strain provided by the invention has stronger lethal activity on second-stage larvae of root-knot nematode, has stronger inhibition effect on incubation of root-knot nematode eggs, has important application value on prevention and control of root-knot nematode diseases of crops, and can be widely applied to preparation of biological agents for preventing and controlling the root-knot nematode diseases.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of microbial pesticides, in particular to a nematode-killing Streptomyces JXSY860 and application thereof. BACKGROUND

[0002] Plant parasitic nematodes cause plant infectious diseases more than bacteria and viruses in the four pathogens, and only second to fungi. Root-knot nematodes are an important group among them.

[0003] Root-knot nematodes occur widely in the world, have a wide host range, multiple transmission routes, and cause heavy damage. They often cause crop yield loss of 10-30%, and can reach more than 70% in severe cases. In recent years, due to the expansion of facility vegetable planting area, the increase of multiple cropping index, and the single planting of products, plant parasitic nematode diseases have been increasing, which seriously affects the yield and quality of crops and brings great threat to crop production.

[0004] Chemical control is the most widely used method for nematode control at present, mainly through the use of synthetic chemical insecticides such as thiazolide, fluopyram, fluazamamide, and triflumpyridine amine. However, chemical pesticides not only have high toxicity and high cost, but also may have negative effects on beneficial organisms in the soil, and long-term root irrigation with pesticides can also easily cause nematode resistance. Therefore, it is urgent to develop safer and more effective control measures. Biological control as a safe and effective means has been widely used in crop nematode control, which can not only reduce the occurrence of crop nematode disease to a certain extent, but also improve the soil and product quality in the long term, providing protection for sustainable agricultural development. SUMMARY

[0005] The present application aims to provide a nematode-killing Streptomyces JXSY860 and application thereof to solve the problems existing in the prior art. The Streptomyces turgidiscabies JXSY860 has stronger lethal activity on the second instar larvae of root-knot nematodes and stronger inhibitory effect on the hatching of root-knot nematode eggs than other existing strains, and has important application value for the prevention and control of crop root-knot nematode disease.

[0006] To achieve the above-mentioned purpose, the present application provides the following solutions.

[0007] The present application provides a Streptomyces turgidiscabies JXSY860, which was preserved in the Guangdong Microbial Culture Collection Center on August 8, 2024, with a preservation number of GDMCC No: 64998 and a preservation address of 5th Floor, No. 59 Building, Institute of Microbiology, Guangdong Academy of Sciences, 100 Middle Martyrs Road, Guangzhou, China.

[0008] The application further provides a fermentation liquor of the Streptomyces tumescens JXSY860, wherein the fermentation liquor is obtained by centrifugation and filtration of a bacterial suspension of the Streptomyces tumescens JXSY860 cultured.

[0009] Optionally, the Streptomyces tumescens JXSY860 is inoculated into a Gao No. 1 liquid medium for culture, wherein the Gao No. 1 liquid medium is soluble starch 20 g, KNO3 1 g, NaCl 0.5 g, K2HPO4·3H2O 0.5 g, MgSO4·7H2O 0.5 g, FeSO4·7H2O 0.01 g and distilled water 1000 mL, and the pH value is adjusted to 7.2-7.4.

[0010] The application further provides a microbial agent containing the Streptomyces tumescens JXSY860 or the fermentation liquor.

[0011] The application further provides application of the Streptomyces tumescens JXSY860 or the fermentation liquor or the microbial agent in prevention and treatment of root-knot nematodes.

[0012] Optionally, the root-knot nematodes include southern root-knot nematodes.

[0013] The application further provides application of the Streptomyces tumescens JXSY860 or the fermentation liquor or the microbial agent in preparation of a medicament for prevention and treatment of root-knot nematodes.

[0014] Optionally, the root-knot nematodes include southern root-knot nematodes.

[0015] The application further provides a medicament for prevention and treatment of root-knot nematodes, wherein an active ingredient of the medicament comprises the Streptomyces tumescens JXSY860 or the fermentation liquor or the microbial agent.

[0016] The application further provides a method for prevention and treatment of root-knot nematodes, and the method comprises the step of treating the root-knot nematodes with the fermentation liquor of the Streptomyces tumescens JXSY860.

[0017] Optionally, the root-knot nematodes include southern root-knot nematodes.

[0018] The application discloses the following technical effects:

[0019] The present application discloses a new strain of Streptomyces turgidiscabies JXSY860, which was preserved in the Guangdong Microbial Culture Collection Center on August 8, 2024, and the preservation number is GDMCC No: 64998. It is verified that the JXSY860 strain provided by the present application has stronger lethal activity on the second instar larvae of root-knot nematodes and stronger inhibitory effect on the hatching of root-knot nematode eggs, has important application value for the prevention and treatment of crop root-knot nematode disease, and can be widely applied in the preparation of biological pesticides for the prevention and treatment of root-knot nematode disease. BRIEF DESCRIPTION OF DRAWINGS

[0020] In order to more clearly illustrate the technical solutions in the embodiments of the present application or the prior art, the drawings needed in the embodiments will be briefly introduced as follows. Obviously, the drawings in the following description are only some embodiments of the present application, and other drawings can also be obtained by those skilled in the art without creative labor.

[0021] Figure 1 It is the morphology of Streptomyces turgidiscabies JXSY860 strain on Gao's No. 1 medium; A: front; B: back;

[0022] Figure 2 It is the photo of Streptomyces turgidiscabies JXSY860 strain under light microscope;

[0023] Figure 3 It is the phylogenetic tree of Streptomyces turgidiscabies JXSY860 strain based on 16S rDNA sequence;

[0024] Figure 4 It is the toxic effect of Streptomyces turgidiscabies JXSY860 strain fermentation broth on the second instar larvae of southern root-knot nematodes; A: sterile Gao's No. 1 liquid medium control; B: JXSY860 strain fermentation broth;

[0025] Figure 5 It is the inhibitory effect of Streptomyces turgidiscabies JXSY860 strain fermentation broth on the hatching of southern root-knot nematode eggs; A: sterile Gao's No. 1 liquid medium control; B: JXSY860 strain fermentation broth;

[0026] Figure 6 It is the photo of using radish seedling method to determine the pathogenicity of Streptomyces turgidiscabies JXSY860 strain. DETAILED DESCRIPTION

[0027] Now, various exemplary embodiments of the present application will be described in detail, which should not be considered as a limitation of the present application, but should be understood as a more detailed description of some aspects, characteristics and embodiments of the present application.

[0028] It is to be understood that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the present application. In addition, where a range of values is provided, it is understood that each intervening value, to the upper and lower limit of the range is also specifically disclosed. Each smaller range between any stated value or intervening value in a stated range and any other stated or intervening value in that stated range is encompassed. The upper and lower limits of these smaller ranges can independently be included or excluded in the range, and are also encompassed by the application, subject to any specifically excluded limit in the stated range. Where the stated range includes one or both of the limits, ranges excluding either or both of the limits are also included.

[0029] Unless otherwise indicated, all technical and scientific terms used herein have the same meaning as those commonly understood by one of ordinary skill in the art to which this application belongs. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present application, the preferred methods and materials are described. All documents mentioned herein are incorporated by reference to disclose and describe the methods and / or materials in connection with which the documents are cited. In case of conflict, the present document will control.

[0030] Various modifications and changes can be made to the specific embodiments of the present application described herein without departing from the scope or spirit of the application. Other embodiments of the application will be apparent to those of ordinary skill in the art from the description and examples presented herein. The description and examples are illustrative of the application and are not intended to limit the scope of the application.

[0031] As used herein, the terms "comprises", "comprising", "includes", "including", "has", "having", "contains", "containing", or variations thereof, are intended to be open-ended terms that mean including, but not limited to.

[0032] Example 1 Isolation, screening and identification of Streptomyces maduraherens JXSY860

[0033] 1. Isolation and screening

[0034] The inventor collected soil at a depth of 0-20 cm in a radish planting area in Shangyou County, Ganzhou City, Jiangxi Province in October 2023, labeled it, put it into a self-sealing bag, and brought it back to the laboratory for storage at 4°C for standby use. Using the dilution plate coating method, 10 g of freshly stored soil sample was weighed, poured into a 250 mL conical flask containing 90 mL of sterile water and 10 glass beads with a diameter of 5 mm, shaken in a 28°C shaking bed for 30 min to mix the microbial cells, and then left to stand for 20-30 s to obtain a 10 -1 dilution liquid, then 0.1 mL of the mixed soil suspension in the conical flask was taken and added to a 2 mL centrifuge tube containing 0.9 mL of sterile water, and mixed by blowing and sucking to obtain a 10 -2 soil suspension dilution liquid. In this way, 10 -3 and 10 -4 soil suspension dilution liquids were obtained. 10 -4The soil suspension diluent was plated, and the solid medium used was Gao's No. 1 medium (soluble starch 20 g, KNO3 1 g, NaCl 0.5 g, K2HPO4·3H2O 0.5 g, MgSO4·7H2O 0.5 g, FeSO4·7H2O 0.01 g, agar 15-20 g, and distilled water 1000 mL, with the pH value adjusted to 7.2-7.4, and the liquid medium without agar), the plated plate was placed on the table for 20-30 min, so that the bacterial liquid was infiltrated into the medium, and then the plate was inverted in a 28°C constant temperature incubator for culture. After actinomycete colonies grew, the actinomycete single colony grown on the plate was transferred into a new medium plate for subculture for 2-3 generations under a sterile environment until the strain was purified.

[0035] By screening, a strain numbered JXSY860 was obtained by using the above method.

[0036] 2. Identification

[0037] (1) Morphological characteristics: after 7 days of culture by the plug method, the JXSY860 strain was cultured on Gao's No. 1 medium, the colony was white, nearly round, the conidial filament was long, curved or slightly curved, and no soluble pigment was produced. The photograph of the JXSY860 strain under an optical microscope is shown in FIG. 1. Figure 2

[0038] (2) Physiological characteristics: the carbon source of the strain JXSY860 was screened, and the results are shown in Table 1: the strain can utilize sucrose, mannitol, raffinose, and lactose, cannot utilize xylose, fructose, and arabinose; can reduce nitrate and hydrolyze starch, cannot liquefy gelatin, and also cannot produce hydrogen sulfide.

[0039] Table 1 Physiological and biochemical characteristics of the strain JXSY860

[0040]

[0041] Note: "+" is a positive result, and "-" is a negative result.

[0042] ​(3) Molecular biological characteristics: The obtained strain JXSY860 was cultured on Gause No. 1 solid medium for 7 days, and then a representative strain colony was picked up with a sterile gun head in an ultraclean bench and placed in a PCR tube. The DNA of the strain was extracted by using a plant DNA extraction kit (universal type). The 16S rRNA gene of the test strain was amplified by using 27F (AGTTTGATCMTGGCTCAG, SEQ ID NO. 1) and 1492R (GGTTACCTTGTTACGACTT, SEQ ID NO. 2) as primers and the test strain as a template. The length of the obtained gene sequence was 1390 bp. The MEGA software was used to construct a phylogenetic tree based on the 16S rDNA sequence by using a neighbor-joining method.

[0043] At the same time, the Blast program was used to compare the nucleic acid homology with the sequences registered in Genebank. The 16S rRNA sequence of the strain had a homology of 99.57% with Streptomyces turgidiscabies, a homology of 98.78% with Streptomyces lacrimifluminis, and a homology of 98.35% with Streptomyces humidus. The analysis result of the phylogenetic tree also showed that the genetic distance of the strain to Streptomyces turgidiscabies was closer (as shown in FIG. 2). Figure 3

[0044] In combination with the colony morphological characteristics, physiological and biochemical characteristics, and molecular biological characteristics, it was determined that the strain JXSY860 was Streptomyces turgidiscabies JXSY860.

[0045] The Streptomyces turgidiscabies JXSY860 was preserved in the Guangdong Microbial Culture Collection Center on August 8, 2024, with a preservation number of GDMCC No: 64998 and a preservation address of No. 59 Building, 5th Floor, Institute of Microbiology, Guangdong Academy of Sciences, 100 Middle Martyrs Road, Guangzhou.

[0046] Example 2 Preparation of fermentation broth of strain JXSY860

[0047] ​The strain JXSY860 was inoculated under sterile conditions onto the 9 cm Gause No. 1 solid medium in Example 1, and grown at 25°C in the dark for about 5 days to obtain the use. A single colony well grown on the plate was picked up in a centrifuge tube containing 30 mL of Gause No. 1 liquid medium, and cultured at 200 rpm for 120 h to obtain a bacterial suspension. The supernatant was obtained by centrifugation at 10,000 rpm for 10 min, and filtered through a 0.22 μm bacterial filter to obtain the fermentation broth of the strain JXSY860. At the same time, the sterile liquid Gause No. 1 medium (without the strain) was centrifuged at 10,000 rpm for 10 min, filtered through a 0.22 μm filter, and stored at 4°C for use as a control.

[0048] Example 3 Determination of the effect of the fermentation broth of the strain JXSY860 on the root-knot nematode

[0049] (1) Cultivation of the root-knot nematode

[0050] The tomato root system infected with the southern root-knot nematode was collected, washed with water, and the egg capsules were carefully removed. The egg capsules were sterilized in a 1% sodium hypochlorite solution for 3 min, washed with sterile water for 3 times, and placed in a culture dish containing a small amount of sterile water, and incubated at 25°C. The newly hatched 2nd instar larvae of the southern root-knot nematode were collected every 24 h, and stored in a 4°C refrigerator for use.

[0051] (2) Comparative strains

[0052] Streptomyces enissocaesilis: a root-knot nematode biocontrol strain purchased from the China General Microbiological Culture Collection Center, with the accession number CGMCC No: 4.1586.

[0053] Strains 1583-1 and 1324-2: other Streptomyces strains found by the inventors during the screening of the strain JXSY860 of S. maduroides.

[0054] (3) Determination of the nematocidal effect

[0055] 500 μL of the fermentation broth of strain JXSY860 prepared in Example 2 was added to 24-well cell culture plates, along with 500 μL of a suspension of 60-100 southern root-knot nematodes for virulence testing. The currently available root-knot nematode biocontrol strain Streptomyces lilacinus and strains 1583-1 and 1324-2, screened using the same method, were used as control strains. Sterile water and sterile Gao's broth medium (G-CK) were used as controls. Each treatment was repeated four times. After 24 hours, the cells were placed in a 25°C incubator. Nematode mortality was recorded and confirmed by needle contact to determine whether the nematodes were alive or dead. Corrected mortality was calculated. Nematode lethality = dead nematodes / total number of test nematodes × 100%; corrected mortality (%) = (death rate of treated nematodes - death rate of control nematodes) / (1 - death rate of control nematodes) × 100%. Dead nematodes are those that did not move under physical stimulation. Four biological replicates were performed at each time point.

[0056] The results are as follows Figure 4 As shown in Table 3, after 24 hours of treatment with the fermentation broth of strain JXSY860, the nematodes were essentially frozen in death, unable to move even when pricked, with a corrected mortality rate of 92.37%. This compares to only 27.35%, 41.4%, and 47.70% for the control strains 1583-1, 1324-2, and Streptomyces lilacinus, respectively (Table 3). This indicates that the fermentation broth of strain JXSY860 is significantly more toxic to second-instar larvae of the southern root-knot nematode than the control strains.

[0057] Table 3 Effects of each group on the second-instar larvae of southern root-knot nematode

[0058]

[0059]

[0060] Note: The average mortality rate is expressed as mean ± standard error. Different letters in the same column indicate significant differences among treatments (P<0.05).

[0061] Example 4 Determination of the effect of the fermentation broth of strain JXSY860 on the hatching of root-knot nematode eggs

[0062] The fermentation broth of 500 μL of the strain JXSY860 prepared in Example 2 was added to a 2 mL centrifuge tube, and 500 μL of sterile water was added to make the dilution of the bacterial suspension 2x. Two sterilized egg capsules of similar color and size were added to each tube. The fermentation broth of the same volume of S. violaceus, the 1583-1 strain and the 1324-2 strain were used as a comparison, and the same volume of sterile Gao's No. 1 liquid medium and sterile water were used as a control. Each treatment was repeated 3 times, and the number of hatched nematodes and un-hatched eggs was counted after 5 days in a 25°C incubator. The hatching rate and hatching inhibition rate were calculated. The hatching rate = (the number of hatched second instar larvae x 100) / the number of starting mature eggs x 100%, and the hatching inhibition rate (%) = (the control hatching rate - the treatment hatching rate) / the control hatching rate x 100%.

[0063] The results are shown in Table 4 and Figure 5 The fermentation broth of the strain JXSY860 significantly inhibited the hatching of the eggs of the root-knot nematode, with an inhibition rate of 85.71%. In comparison, the inhibition rates of the fermentation broth of the strains 1583-1, 1324-2 and S. violaceus were only 22.86%, 25.80% and 53.55%, respectively. This indicates that the fermentation broth of the strain JXSY860 has a stronger inhibitory effect on the hatching of the eggs of the root-knot nematode, and the inhibitory effect is significantly better than that of the fermentation broth of the strains 1583-1, 1324-2 and S. violaceus.

[0064] Table 4 Inhibitory effect of each group on the hatching of the eggs of the southern root-knot nematode

[0065]

[0066] Note: The inhibition rate is expressed as the mean ± standard error, and the same column with different letters indicates a significant difference (P < 0.05) between treatments.

[0067] Example 5 Determination of the potting control effect of the fermentation broth of the strain JXSY860 on the southern root-knot nematode

[0068] Tomato seeds were surface sterilized, soaked in sterile water for 12 h, and then planted in plastic pots containing sterilized substrate. After 15 days, they were transplanted into a pot, with 1 seed per pot, and watered regularly. When they grew to two leaves and one heart, about 250 freshly hatched root-knot nematode J2s were added. After 12 h, the fermentation broth of the strain 2x dilution, the abamectin technical dilution (1 ppm·plant-1) and the sterile Gao's No. 1 liquid medium 2x dilution were added by root irrigation, with 30 plants per treatment, 50 mL per plant. The fermentation broth of the strain 2x dilution was added again by root irrigation after one week, with 50 mL per plant. -1 ) and the sterile Gao's No. 1 liquid medium 2x dilution were added by root irrigation, with 30 plants per treatment, 50 mL per plant. The fermentation broth of the strain 2x dilution was added again by root irrigation after one week, with 50 mL per plant. 1) and sterile Gao's No. 1 liquid medium. Routine management was performed during tomato growth. Sixty days after nematode inoculation, the roots were carefully cleaned and the fresh root weight, stem fresh weight, stem diameter, and plant height were measured. The roots were graded based on the proportion of knotted roots to the total number of roots, and the disease index and control efficacy were calculated.

[0069] The grading standards for root-knot nematode disease are as follows: Level 0: no root knots; Level 1: the root knot ratio is <25%; Level 3: the root knot ratio is 25%-50%; Level 5: the root knot ratio is 50%-75%; Level 7: the root knot ratio is more than 75%.

[0070] Disease index = ∑ (representative value of each level × number of diseased plants at this level) / (total number of plants surveyed × highest level representative value) × 100; control effect = [1 - (treatment disease index / control disease index)] × 100%.

[0071] Results: The efficacy of the fermentation liquid of strain JXSY860 against southern root-knot nematodes in potted plants was studied under greenhouse conditions. Application of the fermentation liquid of strain JXSY860 and avermectin significantly reduced the number of root knots in tomatoes by 31.27% and 56.74%, respectively, and achieved control efficacy of 44.75% and 57.78%, respectively, against root-knot nematodes. Furthermore, the stem diameter of tomato plants treated with the fermentation liquid of strain JXSY860 was significantly reduced by 8.27% compared with the control (CK) and the plant height was significantly increased by 25.12%. Root and stem weights did not change significantly compared with the CK. Abamectin-treated tomato plants had significantly higher root weight, stem weight, stem diameter, and plant height than the CK and fermentation liquid of strain JXSY860. These results indicate that the fermentation liquid of strain JXSY860 not only has a good control effect on tomato root-knot nematodes but also has a certain effect on promoting plant height, although the effect is slightly lower than that of avermectin (Table 5).

[0072] Table 5 Control effect of fermentation liquid of strain JXSY860 on southern root-knot nematode in potted tomatoes

[0073]

[0074] Note: Different letters in the same row indicate significant differences among treatments (P<0.05).

[0075] Example 6 Determination of pathogenicity of strain JXSY860

[0076] Radish seedling detection method: radish seeds are first soaked in 0.26% NaClO solution for about 10 min, washed with sterile water for 3 times, dried, placed in a sterile filter paper moistened culture dish for 24 h, and the seeds with consistent germination are selected and placed in a test tube containing 5 mL of 1% water agar medium, 1 seed per tube, inoculated with 200 μL of JXSY860 bacterial suspension and sterile Gao's No. 1 liquid medium (control) respectively, 20 tubes per treatment, placed in a 25°C light incubator, light time is 16 h / d, and the height of seedlings is measured after 7 d of inoculation, and the inhibition rate of each treatment is used as the pathogenicity index. Inhibition rate = (1- average value of inoculated seedling length / average value of control seedling length) x 100%.

[0077] Results: the growth of carrot seedlings inoculated with strain JXSY860 bacterial suspension has no significant difference compared with the control (see Table 6). Figure 6 As can be seen from Table 6, strain JXSY860 bacterial suspension has no inhibitory effect on seedling growth. This shows that the screened strain JXSY860 has no pathogenicity.

[0078] Table 6 Inhibition of JXSY860 bacterial suspension on the growth of carrot seedlings

[0079]

[0080] Note: 0d is the length of carrot seedlings before inoculation.

[0081] According to the above experimental results, it can be seen that the fermentation broth of Streptomyces turgidiscabies JXSY860 provided by the present application not only has strong toxic effect on second instar larvae of root-knot nematodes, but also can inhibit egg hatching to a great extent, and the strain has no pathogenicity, and has important application value for the prevention and control of crop root-knot nematode disease.

[0082] The above examples only describe the preferred modes of the present application, and do not limit the scope of the present application. Without departing from the design spirit of the present application, various modifications and improvements of the technical solutions of the present application made by those skilled in the art shall fall within the protection scope determined by the claims of the present application.

Claims

1. A strain of Streptomyces turgidiscabies JXSY860, characterized in that: It was deposited in Guangdong Provincial Microbiological Culture Collection Center on August 8, 2024, with the deposit number GDMCC No: 64998.

2. The fermentation liquid of Streptomyces ulmoides JXSY860 according to claim 1, characterized in that The bacterial suspension is obtained by culturing Streptomyces scabris JXSY860, and the bacterial suspension is obtained by centrifugation and filtration.

3. A microbial agent, characterized in that: Contains the Streptomyces scabies JXSY860 according to claim 1 or the fermentation liquid according to claim 2.

4. Use of the Streptomyces scabies JXSY860 according to claim 1, the fermentation liquid according to claim 2, or the microbial agent according to claim 3 in preventing and controlling root-knot nematodes.

5. The use according to claim 4, characterized in that The root-knot nematodes include the incognita root-knot nematode.

6. Use of the Streptomyces edema JXSY860 according to claim 1, the fermentation liquid according to claim 2, or the microbial agent according to claim 3 in preparing a medicament for controlling root-knot nematodes.

7. The use according to claim 6, characterized in that The root-knot nematodes include the incognita root-knot nematode.

8. A medicament for preventing and treating root-knot nematode diseases, characterized in that: The active ingredient of the medicament includes the Streptomyces scabies JXSY860 according to claim 1 or the fermentation broth according to claim 2 or the microbial agent according to claim 3.

9. A method for controlling root-knot nematodes, characterized in that: The method comprises the step of treating the root-knot nematodes by using the fermentation liquid of the Streptomyces scabies JXSY860 according to claim 1.

10. The method according to claim 9, wherein The root-knot nematodes include the incognita root-knot nematode.

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