Streptomyces virginiae GZ2 and application thereof in prevention and control of plant parasitic nematodes

Abstract

The invention belongs to the technical field of biological prevention and control of plant parasitic nematodes, and particularly relates to streptomyces virginiae GZ2 and application thereof in prevention and control of plant parasitic nematodes, the streptomyces virginiae GZ2 is preserved in the China Center for Type Culture Collection, and the preservation number is CCTCC M 2024975. The streptomyces virginiae GZ2 fermentation liquor screened by the invention has relatively strong lethal activity on various plant parasitic nematodes, and the corrected death rates of the streptomyces virginiae GZ2 fermentation liquor on meloidogyne incognita, pine wood nematodes, aphelenchoides besseyi and sweet potato stem nematodes are 88.2%, 88.8%, 88.2% and 78.3% respectively. Wherein the corrected death rate of the second-instar larvae of the meloidogyne incognita within 48 hours reaches 99.3%.

Description

Technical Field

[0001] The invention relates to the technical field of biological control of plant parasitic nematodes, and in particular to Streptomyces virginica GZ2 and application thereof in the control of plant parasitic nematodes. Background Art

[0002] Plant parasitic nematodes are an important class of plant pathogens that are widely distributed around the world and have strong adaptability to the environment. They establish long-term, stable parasitic relationships with host plants, causing serious harm to food, horticultural crops and forest plants, causing global economic losses of up to $100 billion each year.

[0003] Biological control of plant parasitic nematodes is one of the important aspects of agricultural plant disease control. Among them, root-knot nematodes are soil-borne diseases with a wide host range and serious damage. They are the most important and difficult to control plant parasitic nematodes.

[0004] At present, the control of plant root-knot nematodes mainly relies on chemical pesticides. Chemical nematicides are extremely toxic and have large residues in soil, water and agricultural products, seriously polluting the environment and food and threatening human health. With the increasing attention paid to food safety and environmental issues, most highly toxic chemical nematicides have been banned or restricted in use. Currently, only a few nematicide products such as avermectin and thiazothion support market demand.

[0005] The root-knot nematode biocontrol microorganisms that have been discovered and applied in production are mainly fungi and bacteria, such as Paecilomyces lilacinus and Pulcinia chlamydosus. Among them, Paecilomyces lilacinus has been industrialized and widely used in root-knot nematode control. Pulcinia chlamydosus is a parasite of root-knot nematode eggs and female worms, and its commercial preparations have good control effects on vegetable root-knot nematodes. Research on bacterial nematode biocontrol preparations mainly focuses on Bacillus amyloliquefaciens, Bacillus thuringiensis and Pseudomonas fluorescens. There are few types of existing root-knot nematode biocontrol fungi and bacterial preparations, and the control effect is unstable, which makes it difficult to effectively solve the problem of root-knot nematode infestation.

[0006] Therefore, it is necessary to provide Streptomyces virginica GZ2 and its application in the control of plant parasitic nematodes to solve the above technical problems. Summary of the invention

[0007] In order to solve the above technical problems, the present invention provides Streptomyces virginica GZ2 and its application in controlling plant parasitic nematodes.

[0008] The present invention provides Streptomyces virginica GZ2 and its application in controlling plant parasitic nematodes.

[0009] Streptomyces virginiae, specifically Streptomyces virginiae GZ2, is deposited in China Center for Type Culture Collection with the accession number CCTCC M 2024975. The accession number in NCBI is PQ796982.

[0010] (II) Application of Streptomyces virginica

[0011] (1) Application of Streptomyces virginiae GZ2 in the control of root-knot nematodes.

[0012] (2) Application of Streptomyces virginiae GZ2 in the control of pine wood nematodes.

[0013] (3) Application of Streptomyces virginiae GZ2 in the control of rice stem nematode.

[0014] (4) Application of Streptomyces virginiae GZ2 in the control of sweet potato stem nematodes.

[0015] (5) Application of Streptomyces virginiae GZ2 in promoting plant growth

[0016] Compared with the related art, the Streptomyces virginica GZ2 provided by the present invention and its application in the control of plant parasitic nematodes have the following beneficial effects:

[0017] 1. The fermentation liquid of Streptomyces virginiana GZ2 screened by the present invention has strong lethal activity against a variety of plant parasitic nematodes, and the corrected mortality rates against southern root-knot nematodes, pine wood nematodes, rice stem nematodes and sweet potato stem nematodes are 88.2%, 88.8%, 88.2% and 78.3% respectively. Among them, the corrected mortality rate against the second-instar larvae of southern root-knot nematodes in 48 hours reaches 99.3%.

[0018] 2. The Virginia Streptomyces GZ2 screened by the present invention can synthesize two plant growth stimulants and growth-promoting substances. The growth stimulant is indoleacetic acid (IAA), and the growth-promoting substance is siderophore, both of which can significantly promote the growth of crops. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1 A schematic diagram of the position of the Virginia Streptomyces GZ2 provided by the present invention and the strain GZ2 used in the control of plant parasitic nematodes on the evolutionary tree;

[0020] Figure 2A schematic diagram of the death state of nematodes after being treated with the fermentation supernatant of Streptomyces virginiae GZ2 provided by the present invention and its application in the control of plant parasitic nematodes for 48 hours. DETAILED DESCRIPTION

[0021] The technical solution of the present invention will be described clearly and completely below in conjunction with the accompanying drawings. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0022] Embodiment 1

[0023] In view of the problems existing in the prior art, the purpose of the present invention is to screen out a multifunctional Streptomyces that has strong insecticidal activity against plant parasitic nematodes and can promote plant growth, and at the same time provide the application of the bacteria in preventing and controlling plant parasitic nematodes and promoting growth.

[0024] In order to achieve the above object, the present invention adopts the following technical solutions to achieve it.

[0025] The embodiments of the present invention will be described in detail below with reference to examples, but those skilled in the art will understand that the following examples are only used to illustrate the present invention and should not be construed as limiting the scope of the present invention.

[0026] Actinomycetes are the main producers of antibiotics. Among the existing antibiotics, about 70% are produced by microorganisms; among the antibiotics produced by microorganisms, about 70% are produced by actinomycetes; and among the antibiotics produced by actinomycetes, about 70% are produced by Streptomyces. Therefore, Streptomyces is the main producer of antibiotics and an important target for screening of biocontrol bacteria for root-knot nematodes. Streptomyces is the largest genus in the order Actinomycetales, with more than 500 effective species. The antibiotics synthesized by Streptomyces are mainly used in medicine and have strong antibacterial and antifungal functions, but Streptomyces are not widely used in agriculture, especially in the control of plant parasitic nematodes, which has not attracted widespread attention. In addition to being able to produce a variety of antibiotics, Streptomyces also has an important feature: it can produce a large number of spores with extremely strong stress resistance and can survive for a long time under dry conditions. This feature gives Streptomyces important commercial value: live bacterial preparations have a long shelf life.

[0027] The present invention provides a strain of Streptomyces, specifically Streptomyces virginica GZ2, which was deposited in the China Center for Type Culture Collection on May 17, 2024, with a deposit number of CCTCC M 2024975; the accession number in NCBI is PQ796982.

[0028] The test materials used in this embodiment are as follows:

[0029] (1) Streptomyces virginica GZ2: isolated and screened from the soil of Guanshan Lake District, Guiyang City, Guizhou Province.

[0030] (2) Tomato variety: Hezuo 903, produced by Shanghai Hongqiao Tianlong Seed Co., Ltd., is a variety that is sensitive to root-knot nematode infection and susceptible to disease.

[0031] Example 1 Identification of test strains

[0032] 1. Morphological characteristics:

[0033] The screened actinomycete GZ2 with high nematicidal activity was inoculated on ISP1, ISP2, ISP3, ISP4, ISP5, ISP7, Gao's medium No. 1, potato dextrose agar medium, Czapek medium and nutrient agar medium using the three-zone streak method; single colonies were obtained by the dilution plate smear method. The inoculated plates were inverted and cultured at 28°C for 21 days, and the colony morphology was observed and described: size, edge condition, degree of protrusion, surface gloss, colony color and other characteristics (Table 1).

[0034] Table 1. Morphological characteristics of Streptomyces virginica GZ2 on 10 culture media

[0035] Culture medium Growth status Colony morphology Soluble Color Aerial hyphae ISP1 ++ The colony is small, not easy to pick up, with white spots in the middle, and produces spores yellow Underdeveloped, yellow ISP2 ++++ The colony is large, easy to pick up, and has white spots in the middle yellow Developed, white ISP3 ++++ The colony is large, easy to pick up, and has well-developed spores Light yellow Very developed, gray-pink ISP4 +++ The colonies are small and round, easy to pick up, and the spores are not well developed Light yellow More developed, white ISP5 +++ The colonies are small and round and easy to pick up Light pink White ISP7 ++ The colony is smaller, easier to pick up, and the spores are more developed Light yellow More developed, light yellow PDA ++++ The colonies are small, easy to pick up, grow vigorously, and produce a large number of spores tan Well-developed, lavender-gray GA ++ The colony is large, easy to pick up, and has a light yellow spot in the middle Light pink More developed, pink-gray CDA +++ The colonies are small and round, easy to pick up, and produce spores Light pink Well-developed, lavender-gray NA + The colony is very small and difficult to pick up, with white spots in the middle - Underdeveloped, white

[0036] Note: “++++”, “+++”, “++” and “+” represent good, relatively good, fair and poor growth respectively.

[0037] 2. Physiological and biochemical characteristics

[0038] The test strains were inoculated or plated in different physiological and biochemical culture media, and the growth of the actinomycetes in the culture media was observed and recorded (Table 2).

[0039] Table 2. Physiological and biochemical characteristics of Streptomyces virginica GZ2

[0040] Biochemical function Reaction / content Carbon source utilization reaction Milk coagulation and peptization + glucose + Gelatin liquefaction + sucrose + Hydrogen sulfide production + maltose + Melanin production + D-Mannitol + Nitrogen fixation + Soluble starch + Siderophilase + D-Fructose + Auxin content (μg / mL) 23.6 D-Sorbitol + Phosphate Dissolution - Cellulose decomposition -

[0041] 3. Molecular identification of actinomycetes

[0042] 16S rDNA gene fragment amplification and sequencing: The mycelia of strain GZ2 cultured on Gao's medium No. 1 were scraped with a sterile blade and collected in a sterile 1.5 mL centrifuge tube. After rapid freezing into powder with liquid nitrogen, 200 μL Elution Buffer was added, followed by 20 μL of 20 mg / mL proteinase K solution. After oscillation and mixing, the cells were placed in a 56°C water bath for 1 h to lyse the cells. According to the instructions of the PCR purification kit Cycle-pure kit (200), the purified DNA of the test actinomycetes was extracted and stored in a -20°C freezer.

[0043] The purified DNA of the test bacteria was used as a template, and the 16SrDNA gene fragment was amplified using the universal primer pair 27F (5'-AGA GTT TGATCC TGG CTCAG-3') and 1492R (5'-TAC GAC TTAACC CCAATC GC-3'). The primers were synthesized by Nanjing Qingke Biotechnology Co., Ltd. The PCR amplification system was 25 μL: 1 μL of actinomycete DNA template, 1 μL of upstream and downstream primers, 12.5 μL of 2×GS Taq polymerase, and 9.5 μL of ddH2O. The amplification reaction conditions were: 95℃ pre-denaturation for 10 min; 95℃ denaturation for 1 min, 56℃ annealing for 1 min, 72℃ extension for 1 min, 35 cycles; 72℃ total extension for 10 min. The amplified product was detected by 1.0% agarose gel electrophoresis at a stable voltage of 110V for 25min, the gel was stained with 1.0% ethidium bromide (EB) for 20min, and observed and photographed under a UV gel imager. The target band size was about 1400-1500bp. The remaining PCR product was sent to Shanghai Shenggong Biological Company for purification and sequencing.

[0044] Phylogenetic analysis of 16S rDNA gene: After the sequences obtained by sequencing were spliced, the sequences were compared and analyzed using BLAST search software in NCBI. Due to the small differences between bacterial species, it is difficult to obtain accurate molecular identification results based solely on 16S rDNA gene sequence analysis. Phylogenetic trees were constructed based on 16S rDNA gene sequences and whole genome sequences, respectively. For the 16S rDNA evolutionary tree, bacterial communities with distant genetic relationships were downloaded as outgroups, and multiple sequence alignments were performed using the MAFFT (https: / / mafft.cbrc.jp / alignment / server) online platform. The fasta files generated after the alignment were converted to the format using ALTER, and a maximum likelihood (RAxML) evolutionary tree was constructed in IQTree, and 1000 bootstrap operations were performed. The larger the Bootstrap value (BS), the higher the confidence level (Stamatakis et al., 2014). The maximum likelihood method was used to construct the 16S rDNA gene phylogenetic tree of the test strain ( Figure 1 ).

[0045] From the evolutionary tree, it can be seen that strain GZ2 is clustered with Streptomyces virginiae on the same branch, and the strain can be identified as Streptomyces virginiae.

[0046] 16S rDNA sequence of GZ2:

[0047]

[0048] Example 2 Nematicidal Activity of Streptomyces virginica

[0049] 1. Preparation of Fermentation Broth of Streptomyces virginica GZ2

[0050] The Virginia Streptomyces strain preserved in the slant Gower's No. 1 medium was streaked on a Gower's No. 1 solid medium plate, and after culturing at 28°C for 7 days, a single colony was picked and inoculated into a test tube containing 5 mL of Gower's No. 1 liquid medium, and cultured at 28°C in a shaker at 180 rpm for 7 days to obtain Virginia Streptomyces fermentation broth; the fermentation broth was centrifuged at 12000 rpm for 3 minutes to obtain Virginia Streptomyces fermentation supernatant.

[0051] 2. Determination of nematicidal activity of GZ2

[0052] In a 96-well cell culture plate, 80 μL of the supernatant of Streptomyces virginica fermentation and 20 μL of the test nematode suspension (containing 20-30 nematodes) were placed in one well. The control treatment was 80 μL of liquid Gao's No. 1 medium and 20 μL of nematode suspension. Each treatment was repeated in 3 wells. After counting the nematodes in each well under a stereomicroscope, the 96-well plate was placed in a 25°C incubator. The activity of the nematodes treated with Streptomyces virginica and the control treatment was observed at 24h and 48h, and the number of nematode deaths was counted. The nematode body was considered dead if it was stiff ( Figure 2 ). Calculate the nematode mortality of each treatment and use formula (1) to calculate the corrected mortality.

[0053] (1)

[0054] Table 3. Corrected mortality of five nematodes by fermentation broth of Streptomyces virginica GZ2 at 48 hours (%)

[0055]

[0056] 3. Comparison of the insecticidal activity of Streptomyces virginica GZ2 and chemical nematicides against southern root-knot nematodes

[0057] The southern root-knot nematode subcultured in our laboratory was used as the target to determine the difference in anti-insect activity between GZ2 and chemical nematicides. The roots of diseased tomato plants with a large number of egg masses were washed, and the egg masses were picked up with pointed tweezers, surface disinfected with 1% sodium hypochlorite for 3 minutes, washed with sterile water 5 times, and placed in a 28℃ incubator for incubation. The second-instar larvae (J2) suspension of the root-knot nematode were collected and diluted to 1000 / mL with sterile water.

[0058] In a 96-well cell culture plate, 80 μL of the fermentation supernatant of the test bacteria and 20 μL of the southern root-knot nematode J2 suspension (containing 20-30 nematodes) were placed in one well. The control treatment (CK) was 80 μL of liquid Gao's medium No. 1 and 20 μL of nematode suspension. Each treatment was repeated in 3 wells. After counting the nematodes in each well under a stereomicroscope, the 96-well plate was placed in a 25°C incubator. The activity of the nematodes treated with Virginia Streptomyces GZ2 and the control treatment was observed at 24 h and 48 h, and the number of nematode deaths was counted. In order to avoid statistical errors caused by pseudo-death of nematodes, 1N NaOH sodium hydroxide can be added to observe whether the nematodes are revived. If the worm body is wriggling, it is considered alive, and if the worm body is stiff, it is considered dead. The nematode mortality rate of each treatment was calculated, and the corrected mortality rate was calculated using formula (1). The disease index and control effect were calculated using formulas (2) and (3).

[0059] Table 4. Effects of Streptomyces virginiana GZ2 and chemical nematicides on the reproduction and disease index of southern root-knot nematodes

[0060]

[0061] Note: 1. J2 is the second instar larva of root-knot nematode. 2. Different lowercase letters in the same column indicate significant differences at P < 0.05.

[0062] (2)

[0063] (3)

[0064] Example 3 Determination of the ability of Streptomyces virginica to produce siderophore

[0065] The production of iron carriers was detected by modified ChromeAzurol S (CAS) solid culture medium. Hexadecyl trimethyl ammonium bromide (HDTMA) forms a bright blue complex with iron ions. When the iron ions in the culture medium are taken away by the siderophore secreted by the strain, the area around the colony will turn orange. A single colony with good growth was selected and inoculated on the modified CAS solid culture medium, cultured at 28°C for 7 days, and the growth of the test bacteria on the culture medium was observed. If an orange-yellow circle appears around the colony, it means that the test bacteria produces siderophore enzymes, and the inner and outer diameters of the orange-yellow circle are recorded. The test results show that Streptomyces virginica produces siderophore enzymes (Table 2).

[0066] Example 4 Determination of the ability of Streptomyces virginica to produce auxin

[0067] The ability to produce auxin (IAA) was determined by the Salkowski colorimetric method.

[0068] Auxin production: The tested Streptomyces virginica was inoculated into liquid Goulburn medium No. 1 containing 0.5 g / L tryptophan. The culture was carried out at 28°C and 220 rpm for 7 days to obtain a fermentation broth of Streptomyces virginica; the fermentation broth was centrifuged at 12000 rpm for 2 minutes to obtain a fermentation supernatant.

[0069] Preparation of Salkowski colorimetric solution: 1 mL of 0.5 mol / LFeCl3 is mixed evenly with 35% HClO3 and stored away from light. Take 5 new sterilized test tubes, add 1 mL of Virginia Streptomyces fermentation supernatant and 2 mL of Salkowski colorimetric solution, and use blank culture medium as the control group. Place the test tubes at room temperature away from light for 30 minutes and observe the color of the test tubes. If the color turns red, auxin is produced, and its OD value is measured at a wavelength of 530 nm.

[0070] Drawing of standard IAA curve: Weigh 0.01g of pure IAA and dissolve it in a small amount of ethanol, and dilute it to 100mL with distilled water as the mother solution with a concentration of 100μg / mL. Prepare the mother solution into serial dilutions of 0, 5, 10, 15, 20, 25, 30, 35 and 40μg / mL with distilled water. Take 9 sterile test tubes, add 1mL of IAA concentration dilution and 2mL of Salkowski colorimetric solution in turn, mix well, and stand at room temperature in the dark for 30 minutes. Measure the absorbance values ​​of different concentrations of IAA at a wavelength of 530nm under a UV spectrophotometer. Draw a standard curve with IAA concentration (μg / mL) as the horizontal axis and OD value as the vertical axis.

[0071] The results showed that Streptomyces virginica could synthesize auxin, and the IAA content in the fermentation broth was 23.6 μg / mL (Table 2).

[0072] Example 5 Determination of the Growth-Promoting Ability of Streptomyces virginica

[0073] 60 days after inoculation with root-knot nematodes, the tomato plants were carefully pulled out of the pots, the roots were gently washed with tap water, the fresh weight of the stems and leaves and the fresh weight of the roots were weighed, the plant height, stem diameter and root length were measured, photos were taken, and the effect of the fermentation liquid of Streptomyces virginica on the biomass of the tomato plants was calculated.

[0074] Table 5. Growth promoting activity of Streptomyces virginica GZ2 on tomatoes

[0075]

[0076] In summary, the Virginia Streptomyces screened by the present invention can produce compounds with nematicidal activity, and has a strong lethal effect on plant parasitic nematodes, among which the corrected lethality of southern root-knot nematodes, pine wood nematodes, and rice stem nematodes for 48 hours reaches more than 88%, and the lethality of sweet potato stem nematodes is 78%. It has a significant reducing effect on the inhibition of the reproduction and root knot number of southern root-knot nematodes and the disease index, among which the number of egg masses per plant, the number of female worms, the number of root knots and the disease index of GZ2 treatment are reduced by 60%, 69%, 57% and 64% respectively compared with the control, and the control effect on southern root-knot nematodes is 62.9%, and the inhibitory effect is basically consistent with the control effect (70.0%) of the chemical nematicide thiathiophosphine (Table 4).

[0077] The fungus can also produce the auxin indoleacetic acid and iron carriers, which have the function of promoting plant growth, increasing the root length and stem and leaf fresh weight of tomatoes by 19% and 26% respectively compared with the control.

[0078] Although the present invention has been described in detail in this specification by means of general description and specific implementation examples, it is obvious to those skilled in the art that some modifications or improvements can be made to the present invention. Therefore, similar modifications or improvements made without departing from the spirit of the present invention are within the scope of protection claimed by the present invention.

[0079] The above descriptions are merely embodiments of the present invention and are not intended to limit the patent scope of the present invention. Any equivalent structure or equivalent process transformation made using the contents of the present invention specification and drawings, or directly or indirectly applied in other related technical fields, are also included in the patent protection scope of the present invention.

Claims

1. Streptomyces virginica GZ2, characterized in that The Streptomyces virginica GZ2 is deposited in the China Center for Type Culture Collection with the accession number of CCTCC M 2024975.

2. The Streptomyces virginica GZ2 according to claim 1, characterized in that The application of Streptomyces virginica GZ2 in the prevention and treatment of plant parasitic nematodes.

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