High-activity actinomycetes for killing meloidogyne as well as culture method and application thereof

Abstract

The invention discloses a high-activity actinomycete for killing root-knot nematode as well as a culture method and application of the actinomycete. The name of the actinomycetes is Streptomyces griseorubens WZSF-1, the classification name of the actinomycetes is Streptomyces griseorubens, and the preservation number of the actinomycetes in the Guangdong Microbial Culture Collection Center is CDMCC (China General Microbiological Culture Collection Center) 65368. The strain disclosed by the invention has very good insecticidal activity on meloidogyne incognita and elephant bean meloidogyne, and the insecticidal activity can reach 90.22% and 94.76% respectively.

Description

Technical Field

[0001] The invention belongs to the field of biotechnology, and particularly relates to actinomycetes with high activity in killing root-knot nematodes, a cultivation method and application thereof. Background Art

[0002] Root-knot nematodes (Meloidogyne spp.) are obligate plant parasites with a wide host range. Most crops are susceptible to root-knot nematodes, including cash crops, food crops, fruits and vegetables, horticultural plants, and weeds. Root-knot nematodes occur in over 27 provinces in my country, with widespread occurrence in most areas and increasing damage annually. They are one of the most economically important plant parasitic nematodes in Chinese agricultural production. Investigations have shown that the elephant-ear bean root-knot nematode is widespread in Hainan and can parasitize several major cultivated crops in Hainan, including cucurbits, southern medicinal plants, solanaceous crops, and vegetables. In recent years, the nematode has been found on the roots of peppers in Yunnan and Hunan provinces, as well as on the roots of Chinese cabbage in Shaanxi Province, with a trend of gradual expansion from south to north.

[0003] The reproductive period of root-knot nematodes includes three stages: egg, larva and adult. The second-stage juveniles (J2) are the only infective insect stage. They can invade from the apical growth point of the host root system, pass through the intercellular spaces and reach the vascular parenchyma tissue in the cell division zone to feed.

[0004] Like other infectious diseases, my country has consistently advocated a "prevention-first, integrated management" approach to plant protection for root-knot nematode control. Current methods for controlling root-knot nematodes include chemical, physical, biological, agricultural, plant quarantine, and the use of resistant varieties. Biological control, due to its environmental friendliness and safety for humans and animals, has gradually become a hot topic in crop disease control research. Biological control of root-knot nematodes primarily relies on the use of biocontrol microorganisms, including fungi, bacteria, and actinomycetes, with fungi and bacteria being the most commonly used. Currently, Verticillum and Meriaconiospora species have been used for nematode control.

[0005] Currently, the majority of bacteria used for biological control of root-knot nematodes are Bacillus, Pseudomonas, and Pasteurella penetrans. Actinomycetes primarily control root-knot nematodes through antagonism or toxicity. Abamectin, a highly effective nematicidal agent, was isolated from Streptomyces avermitilis. In 1975, the Kitasato Research Institute in Japan isolated avermectin from a secondary product of Streptomyces avermitilis. This product possesses highly effective nematicidal activity. Studies have shown that a concentration of just 0.5 μg / L can kill root-knot nematodes within 12 hours. Abamectin is currently widely used as a nematicide, insecticide, and acaricide on fruits, vegetables, and grain crops. Treatment of cucumber and cotton seeds with avermectin can reduce root-knot nematode infection by 20%-80%. Nematicidal bacteria are primarily found in the genus Streptomyces. Ruanpanun isolated 83 strains whose metabolites exhibited nematicidal activity from soil samples in Thailand, of which 97.6% were Streptomyces. Jin Han et al. incubated the fermentation supernatant of Streptomyces nigricans with suspensions of second-instar larvae and eggs of root-knot nematodes. They determined the toxicity and inhibitory effects of the fermentation supernatant against second-instar larvae and eggs of root-knot nematodes. The results showed that after 24 hours of incubation, the supernatant had an insecticidal activity of 91.21% against second-instar larvae of root-knot nematodes, and after 72 hours of incubation, the inhibitory rate against nematode eggs reached over 80%. Ruanpanun and Chamswarng isolated 25 actinomycete strains from commercial earthworm castings, of which 12 showed significant inhibitory activity against the eggs and larvae of the southern root-knot nematode. All 12 actinomycete strains were identified as Streptomyces.

[0006] Root-knot nematode disease is a global, soil-borne plant disease that causes significant economic losses annually. Traditional nematode control methods are detrimental to sustainable agricultural production due to high costs, high toxicity, the susceptibility of nematodes to developing pesticide resistance, and environmental hazards. Biological control, with its environmentally friendly and safe approaches for humans and animals, has become a hot topic in crop disease control research. Summary of the Invention

[0007] Based on this, this study screened out a line-defense strain with high nematicidal activity and optimized the fermentation culture conditions of the strain.

[0008] Using the second-stage juveniles (J2) of the root-knot nematode Meloidogyne enterolobii as the target nematode, the fermentation supernatants of 42 laboratory-isolated and preserved strains were screened for nematicidal activity using the immersion method. Fourteen strains with nematicidal activity exceeding 50% were identified, of which strain WZSF-1 exhibited the highest nematicidal activity, outperforming the other strains. The nematicidal activity of this strain against second-stage juveniles of the southern root-knot nematode was also tested using the immersion method. The nematicidal activity of the fermentation filtrate, as a stock solution and a two-fold dilution, was found to be 92.23% and 84.21%, respectively. WZSF-1 was identified as Streptomyces griseorhizoides through morphological, physiological, biochemical, and molecular characterization.

[0009] Based on this, the present invention provides an actinomycete that kills root-knot nematodes. The actinomycete is named Streptomyces griseorubens WZSF-1, classified as Streptomyces griseorubens, and its preservation number in Guangdong Provincial Microbial Culture Collection Center is GDMCC No. 65368.

[0010] The application of the actinomycetes in preventing and controlling root-knot nematodes.

[0011] The root-knot nematodes are root-knot nematodes of southern origin and / or root-knot nematodes of elephant ear bean origin.

[0012] The biological product containing the actinomycetes is used for preventing and controlling root-knot nematodes.

[0013] The root-knot nematodes are root-knot nematodes of southern origin and / or root-knot nematodes of elephant ear bean origin.

[0014] The present invention also provides a method for culturing the actinomycetes, which comprises inoculating the actinomycetes into a culture medium containing 1%-3% soluble starch, 1.5% soybean powder, and an initial pH value of 7, wherein the "%" represents the mass percentage.

[0015] The inoculation volume percentage of the inoculation is 5%.

[0016] Wherein, the seed solution for inoculation can be prepared according to the following method:

[0017] The activated single colony was cultured in a culture medium at 28°C for 3 days to obtain seed solution.

[0018] Specifically, a single colony cultured on Gao's No. 1 plate can be picked and cultured in 100 mL of YE medium at 28° C. for 3 days with shaking to obtain seed solution for later use.

[0019] The strain of the invention has good insecticidal activity against southern root-knot nematode and elephant-ear bean root-knot nematode, and the insecticidal activity can reach 90.22% and 94.76% respectively. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Figure 1 The culture characteristics of strain WZSF-1 on different solid culture media.

[0021] Figure 2 These are the SEM characteristics of the biocontrol fungus WZSF-1.

[0022] Figure 3 The phylogenetic tree of Streptomyces WZSF-1 was constructed using the neighbor-proximity method.

[0023] Figure 4 The control effects of different treatments on pepper root-knot nematodes. In the figure, A: root knot index; B: control effect; Note: CK is blank control; AW is 1000-fold dilution of 5% avermectin; F1 is the original solution of Streptomyces WZSF-1 fermentation supernatant; F2 is 5-fold dilution of Streptomyces WZSF-1 fermentation supernatant.

[0024] Figure 5 Figure 3 Effects of different treatments on pepper growth indicators, A: chlorophyll content; B: plant height; C: stem diameter; D: fresh weight of aboveground parts; E: root weight; Note: “*” indicates significant difference at the 0.05 level; “**” indicates significant difference at the 0.01 level; “***” indicates significant difference at the 0.001 level, the same below; AW is a 1000-fold dilution of 5% avermectin; F1 is the fermentation supernatant of Streptomyces WZSF-1; F2 is a 5-fold dilution of the fermentation supernatant of Streptomyces WZSF-1.

[0025] Figure 6 The aboveground growth conditions of peppers are shown in Figure 2. AW is a 1000-fold dilution of 5% avermectin; F1 is a stock solution of the fermentation supernatant of Streptomyces WZSF-1; and F2 is a 5-fold dilution of the fermentation supernatant of Streptomyces WZSF-1.

[0026] Figure 7 The root growth of pepper

[0027] Among them, AW is a 1000-fold dilution of 5% avermectin; F1 is the original solution of the fermentation supernatant of Streptomyces WZSF-1; and F2 is a 5-fold dilution of the fermentation supernatant of Streptomyces WZSF-1.

[0028] Figure 8 PAL activity in pepper leaves under different treatments

[0029] Among them, AW is a 1000-fold dilution of 5% avermectin; F1 is the original solution of the fermentation supernatant of Streptomyces WZSF-1; and F2 is a 5-fold dilution of the fermentation supernatant of Streptomyces WZSF-1.

[0030] Figure 9 CAT enzyme activity in pepper leaves under different treatments

[0031] Among them, AW is a 1000-fold dilution of 5% avermectin; F1 is the original solution of the fermentation supernatant of Streptomyces WZSF-1; and F2 is a 5-fold dilution of the fermentation supernatant of Streptomyces WZSF-1.

[0032] Figure 10 PPO activity in pepper leaves under different treatments

[0033] Among them, AW is a 1000-fold dilution of 5% avermectin; F1 is the original solution of the fermentation supernatant of Streptomyces WZSF-1; and F2 is a 5-fold dilution of the fermentation supernatant of Streptomyces WZSF-1.

[0034] Figure 11 Effects of different treatments on MDA content in pepper leaves

[0035] Among them, AW is a 1000-fold dilution of 5% avermectin; F1 is the original solution of the fermentation supernatant of Streptomyces WZSF-1; and F2 is a 5-fold dilution of the fermentation supernatant of Streptomyces WZSF-1.

[0036] Biomaterial Deposit

[0037] Deposit number: GDMCC No.65368

[0038] Name: Streptomyces griseorubens WZSF-1

[0039] Taxonomic nomenclature: Streptomyces griseorubens

[0040] Depository: Guangdong Provincial Microbiological Culture Collection Center (GDMCC)

[0041] Address: 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou

[0042] Storage time: October 30, 2024

[0043] Survival: Yes. DETAILED DESCRIPTION

[0044] The following examples are provided to facilitate a better understanding of the present invention, but are not intended to limit the present invention. The experimental methods in the following examples, unless otherwise specified, are conventional methods. The materials and reagents used in the following examples, unless otherwise specified, can all be obtained from commercial sources.

[0045] Test strains: The fungi, bacteria, and actinomycetes tested were isolated and identified by the Microbial Resources and Utilization Laboratory of the Institute of Environment and Plant Protection, Chinese Academy of Tropical Agricultural Sciences.

[0046] Nematodes: The tested root-knot nematodes of the elephant ear bean and the root-knot nematode of the southern root were isolated and identified by the Vegetable Disease Research Laboratory of the Institute of Environment and Plant Protection, Chinese Academy of Tropical Agricultural Sciences, and then propagated and preserved in pepper roots.

[0047] Test culture medium

[0048] The test media included LB, PDA, fermentation basal medium, Gao's No. 1, and YE medium. The formulas of each medium are shown in Table 1.

[0049] Table 1 Basic culture medium and formula

[0050]

[0051]

[0052] Example 1. Acquisition and identification of Streptomyces griseorubens WZSF-1

[0053] 1. Screening of Actinomycetes for Root-Knot Nematode Biocontrol

[0054] 1. Obtaining the second-instar larvae of the root-knot nematode

[0055] The roots of peppers that have been colonized by root-knot nematodes are pulled out, the surface soil is washed with clean water, and the root knots are cut into small pieces with scissors; the cut root knots are immersed in clean water, and clean water and sodium hypochlorite solution are added in a volume ratio of 200:1.5, and stirred with a meat grinder for 2-4 times, and then collected in a container and shaken for 5-8 minutes to soften the roots; 100-mesh, 300-mesh, and 500-mesh sieves are stacked from top to bottom, and the softened roots are poured into them. The roots are rinsed with clean water and rubbed by hand to break the root-knot nematode eggs; the 500-mesh sieve is repeatedly rinsed with running water to remove residual sodium hypochlorite until there is no irritating smell, and the root-knot nematode eggs on the 500-mesh sieve are collected and placed on a double layer of lens paper, immersed in clean water and incubated at room temperature. The hatching status can be observed daily under a dissecting microscope; finally, the hatched second-instar larvae are collected and prepared into an adult suspension for later use.

[0056] 2. Preparation of strain fermentation broth

[0057] 1) Seed solution preparation

[0058] 100 μL of actinomycetes stored in glycerol at -80°C was spread on Gao's No. 1 plate and cultured at 28°C for 7-15 days. A single colony was picked and cultured in 100 mL of YE medium at 28°C for 3 days with shaking for later use.

[0059] 2) Fermentation broth preparation

[0060] To prepare actinomycete fermentation broth, inoculate 1% seed solution of each strain into Gao's No. 1 liquid medium and culture at 28°C with shaking for 7 days. Centrifuge the fermentation broth at 10,000 rpm for 10 minutes to obtain the fermentation supernatant, which was stored at 4°C until needed.

[0061] 3. Primary screening and rescreening of nematicidal biocontrol bacteria

[0062] The immersion method was used to determine the activity of biocontrol bacteria against the second-instar larvae of the ear bean root-knot nematode. 900 μL of the actinomycete fermentation supernatant was added to a 24-well plate. 100 μL of a suspension containing approximately 100 second-instar larvae of the elephant ear bean root-knot nematode was then added and mixed. The mixture was then incubated at room temperature for 24 hours. A blank medium was used as a control, and three replicates were set up for each treatment. After treatment, the treated solution was collected into a centrifuge tube and diluted to 8 mL with water. Centrifuged at 2000 rpm for 2 minutes, the supernatant was removed, and the solution was diluted to 8 mL with water and resuspended for 12 hours. Nematodes were counted and counted under a dissecting microscope. Nematodes that were immobile and unresponsive to mechanical touch were considered dead. Mortality was calculated according to the following formula:

[0063] Nematode mortality rate = number of dead nematodes / total number of tested nematodes × 100%

[0064] Corrected mortality rate = (treatment nematode mortality rate - control nematode mortality rate) / (1 - control nematode mortality rate) × 100%

[0065] Strains with high nematicidal activity from the initial screening results were re-fermented in shake flasks to determine their nematicidal activity. Nematicidal activity was measured after 3, 6, 24, and 48 hours of treatment. The fermentation supernatant was diluted 2-fold and 5-fold to determine its activity. This allowed the identification of biocontrol strains with high and stable nematicidal activity.

[0066] The results, shown in Table 2, were obtained by the immersion method, revealing seven actinomycete strains with nematicidal activity against second-instar larvae of the root-knot nematode R. aurantii. Two strains, WZSZJF-2 (9.20) and WZSF-1, exhibited nematicidal activity exceeding 80% in their fermentation supernatant solutions over 24 hours. Their average corrected mortality rates against nematodes were 87.42 ± 1.30% and 88.08 ± 5.65%, respectively. Therefore, WZSZJF-2 (9.20) and WZSF-1 were selected for subsequent rescreening experiments.

[0067] Table 2 Results of preliminary screening of nematode active strains

[0068] strain number Average adjusted mortality rate (%) CK 0.00±0.00f WZSF-9 75.98±2.33ab WZSZJF-1 76.06±6.30ab WZSZJF-2(9.20) 87.42±1.30a WZSZJF-2(11.22) 77.05±9.37ab WZSF-1 88.08±5.65a WZSZJ-7 71.08±7.79b WZSZJ-5 79.29±1.56ab

[0069] Note: Different letters in the table indicate significant differences in nematicidal activity among different strains (P<0.05), the same below.

[0070] Two strains with nematicidal activity greater than 80% in the initial screening results were re-fermented in shake flasks, and their fermentation supernatants were assayed for nematicidal activity at 3, 6, 24, and 48 hours. The fermentation supernatants were also diluted 2-fold and 5-fold to assay their 24-hour nematicidal activity. The results are shown in Tables 3 and 4. The average corrected mortality rates of both strains were greater than 70% when diluted 2-fold, with the average corrected mortality rate of WZSF-1 reaching 81.16±4.04%, and its nematicidal activity was significantly higher than that of the other strains. Even after a 5-fold dilution, the average corrected mortality rate of WZSF-1 remained above 70%, significantly higher than that of the other strains. The fermentation supernatant of each strain was treated with nematodes for 3, 6, 24, and 48 hours. The nematicidal activity of all four strains increased with treatment time. The average corrected mortality rate of WZSF-1 was significantly higher than that of the other strains. At 3 hours of treatment, the average corrected mortality rate of WZSF-1 against second-instar larvae of the ear bean root-knot nematode reached 80.65±4.04%. After 6 hours of treatment, the nematicidal activity of WZSF-1 was not much different from that at 3 hours. After 24 and 48 hours of treatment, the nematicidal activity of WZSZJF-2 (9.20) and WZSF-1 exceeded 80%, but the difference was not significant. Based on the results of the initial and secondary screening, the fermentation supernatant of WZSF-1, its dilutions, and the different treatment times all showed good nematicidal activity. The average nematicidal activity of the initial and secondary screening solutions was 85.94% at 24 hours, thus identifying the strain WZSF-1 with high nematicidal activity.

[0071] Table 3 Effects of different dilution multiples on nematicidal activity

[0072]

[0073] Table 4 Changes in nematocidal activity at different treatment times

[0074]

[0075] 2. Classification and Identification of Biocontrol Bacteria

[0076] 1. Morphological characterization and scanning electron microscopy observation

[0077] The culture medium and its formula required for identification of colony morphology and culture characteristics are shown in Table 5.

[0078] Table 5 Culture characteristics of culture medium

[0079]

[0080] Morphological Observation: Prepare ISP culture media and Gao's No. 1 inverted plates. Inoculate WZSF-1 onto ISP and Gao's No. 1 plates and invert at 28°C for 7-10 days. Observe the colonies of the biocontrol fungus WZSF-1 for color, shape, size, and the presence of soluble pigment according to the "Rapid Identification and Systematic Classification of Actinomycetes" and "Identification Methods for Streptomyces" (Shirlin and Gottlieb 1966).

[0081] Scanning electron microscopy observation: The scanning electron microscopy observation of WZSF-1 was carried out using the insert method (Zhao Bin et al. 2014). After WZSF-1 was streaked on Gao's No. 1 plate, a sterilized cover slip was inserted into the culture medium at a 45° angle perpendicular to the streaking direction and inverted and cultured at 28°C for 5-7 days. The cover slip was then gently pulled out and air-dried. The mycelial growth part was cut into squares of approximately 0.5 cm × 0.5 cm in size. After the sample was dried, it was gold-plated and the mycelium, conidiophores and spore morphology of the strain were observed using a scanning electron microscope.

[0082] The results are shown in Table 6 and Figure 1 As shown, the biocontrol fungus WZSF-1 grew on ISP2, ISP3, ISP4, ISP5, Gao's No. 1, and PDA media, but was unable to grow on Czapek's agar and produced no soluble pigment on any of the media. Aerial hyphae were white on ISP3 and Gao's No. 1, while basal hyphae were pale yellow and white, respectively. On ISP2, both aerial and basal hyphae were yellow. On ISP4, aerial hyphae were off-white, while basal hyphae were white. On ISP5 and PDA, both aerial and basal hyphae were yellow.

[0083] Table 6 Culture characteristics of strain WZSF-1 on different solid media

[0084] culture medium Aerial hyphae Mycelium within the base Soluble pigments Growth status ISP2 light yellow light yellow none ++ ISP3 White light yellow none ++ ISP4 Off-white White none +++ ISP5 yellow yellow none ++ Gao No. 1 White White none +++ Czapek medium - - - - PDA yellow yellow none ++

[0085] Note: “+++” indicates good growth; “++” indicates average growth; “+” indicates weak growth; “-” indicates no growth.

[0086] The mycelial and spore morphology of WZSF-1 were observed by SEM ( Figure 2 The aerial hyphae of actinomycete WZSF-1 are long, flat, compact, and intricately interwoven. The tips of the aerial hyphae differentiate into spiral conidia, which produce many spores in the form of short rods with thorn-like protrusions on the surface.

[0087] 2. Physiological and biochemical identification of biocontrol bacteria

[0088] Table 7: Culture medium and its formula required for identification of physiological and biochemical characteristics of biocontrol bacteria.

[0089] Table 7 Physiological and biochemical characteristics of culture medium

[0090]

[0091]

[0092] 1) Carbon source utilization test

[0093] There are many different carbon-containing compounds in nature, and the ability of a strain to utilize certain carbon-containing compounds as its sole carbon source can serve as a basis for its classification. Using Pugo's basal medium as the carbon source, add 1% D-mannitol, D-fructose, sucrose, glucose, glycerol, L-rhamnose, L-arabinose, and inositol to create different carbon source media. Sterilize the plates and inoculate actinomycetes. A negative control is also included. After culturing at 28°C for 7 days, observe and record the growth of the culture.

[0094] 2) Nitrogen source utilization test

[0095] 1% nitrogen source was added to the nitrogen source utilization basal culture medium with tryptophan, valine, histidine, glycine, proline, leucine, glutamic acid, lysine, asparagine and sole nitrogen source respectively. After sterilization and pouring, the biocontrol bacteria were inoculated. The culture medium without nitrogen source was used as the control. After constant temperature cultivation at 28℃ for 7 days, the growth of the culture medium was observed and recorded.

[0096] 3) pH tolerance test

[0097] Liquid Bennett medium was used as the base medium, and a pH gradient medium ranging from 4.0 to 10.0 was prepared. Actinomycetes were inoculated respectively and cultured on a shaker (28°C, 180 r / min) for 7-14 days. The growth status was observed and recorded.

[0098] 4) NaCl tolerance test

[0099] Different amounts of NaCl were added to Bennett's medium to prepare medium with NaCl concentrations of 1%, 3%, 5%, 7%, 9%, 11%, 13%, and 15%. Actinomycetes were inoculated respectively and cultured in a 28°C incubator for 7 days to observe their growth.

[0100] 5) Phosphorus dissolution and nitrogen fixation experiment

[0101] The biocontrol bacteria were inoculated on phosphate-solubilizing medium and nitrogen-fixing medium respectively, with the uninoculated medium as blank control. After culturing in a constant temperature incubator at 28℃ for 7 days, the formation of transparent circles around the strains was observed. The formation of transparent circles indicated that the strain had the ability to solubilize phosphate and fix nitrogen, otherwise it did not.

[0102] 6) Metabolite experiments

[0103] a. H2S gas generation experiment

[0104] Sulfur-containing organic matter decomposed by the strain forms a black iron sulfide precipitate when it encounters iron salts. Inoculate WZSF-1 onto H2S culture medium and incubate inverted at 28°C for 7 days to observe whether a black precipitate forms around the colony. If so, the strain can decompose sulfur-containing organic matter and produce H2S.

[0105] b. IAA production experiment

[0106] The biocontrol bacteria were inoculated into a king liquid culture medium containing L-tryptophan. After shaking culture at 28°C for 5-7 days, the bacterial liquid was centrifuged to remove the bacteria. 500 μL of the supernatant was aspirated into a test tube. Salkowski reagent (Fe Cl3 4.5 g, dH2O 300 mL, 98% concentrated sulfuric acid 587.7 mL, cooled and adjusted to 1 L) was added. After standing in the dark for 30 minutes, the bacterial liquid was observed to see if it turned red. If so, it was positive, otherwise it was negative.

[0107] c. Urease test

[0108] To verify whether WZSF-1 can produce urease, it was inoculated into urease medium, with no bacteria as blank control, and cultured at 28℃ for 4 days to observe whether the medium turned pink. If the color changed slightly, it indicated that WZSF-1 had the ability to produce urease, otherwise it did not.

[0109] d. Gelatin liquefaction experiment

[0110] This test primarily measures the strain's ability to produce proteases. Gelatin is liquid at temperatures above 25°C and solid at temperatures below 25°C. Protease production by the strain can break down gelatin into small molecules, causing it to liquefy. Otherwise, the gelatin remains solid. Sterilized gelatin culture medium is poured into a test tube and inoculated with WZSF-1 bacteria using the puncture inoculation method. Uninoculated culture medium serves as a blank control. After culturing at 28°C for 7 days, observe the gelatin liquefaction. Before observation, refrigerate the culture medium at 4°C for half an hour. If the experimental group's culture medium liquefies, it indicates that the strain has the ability to produce proteases.

[0111] e. Cellulase production experiment

[0112] Cellulose binds to Congo red to form a red complex. When the strain produces cellulase to degrade cellulose, a clear zone forms around the strain. The size of the clear zone is proportional to the strain's ability to degrade cellulose. WZSF-1 was inoculated onto CMC-Na medium and cultured at 28°C for 7 days to observe whether a clear zone formed.

[0113] f. Starch hydrolysis experiment

[0114] The amylase production capacity of a strain is determined based on the fact that starch turns blue when exposed to iodine. WZSF-1 bacterial culture is inoculated onto a starch medium and cultured at 28°C for 7 days. Lugol's iodine solution is then used to examine the colonies for the formation of a transparent hydrolysis zone. The presence of a hydrolysis zone indicates amylase production, and the size of the hydrolysis zone indicates amylase production.

[0115] g. Nitric acid reduction experiment

[0116] Inoculate the biocontrol bacteria into a nitrate-reducing culture medium and incubate it in a 28°C incubator for one week. A small amount of the liquid is then placed in a test tube and one drop each of indicator solution A and solution B is added. A control is an uninoculated culture medium. A positive result is indicated if the test tube changes color red, while a negative result is indicated if the color remains unchanged.

[0117] The identification results are as follows:

[0118] a. Utilization of sole carbon and nitrogen source

[0119] As shown in Table 8, the results of single carbon source experiments with strain WZSF-1 indicate that the strain cannot utilize L-rhamnose, sucrose, and raffinose, and cannot grow on these three carbon sources. However, it can utilize D-glucose, D-fructose, D-mannitol, L-arabinose, inositol, and glycerol, with optimal growth on glucose and rhamnose. Results from single nitrogen source experiments indicate that the biocontrol strain WZSF-1 can utilize tryptophan, valine, histidine, glycine, proline, leucine, and asparagine, but cannot utilize glutamate and lysine.

[0120] Table 8. Carbon and nitrogen source utilization of strain WZSF-1

[0121] Carbon source utilization result Nitrogen source utilization result D-glucose + Tryptophan + D-fructose + Valine + D-Mannitol + Histidine + L-arabinose + glutamate - L-Rhamnose - Glycine + Raffinose - Lysine - sucrose - Proline + Inositol + Leucine + glycerin + Asparagine +

[0122] Note: “+” means positive; “-” means negative

[0123] b. Enzymatic characteristics and physiological properties

[0124] As shown in Table 9, the biocontrol bacterium WZSF-1 has the ability to produce cellulase, protease, and amylase, and has the ability to produce IAA and siderophore, but does not produce urease and hydrogen sulfide, and does not have the ability to solubilize phosphorus, fix nitrogen, or reduce nitrate; it can grow in the pH range of 5 to 10, and grows best in the pH range of 7-8; it can grow well in the salt concentration range of 1% to 13%, and grows best at a salt concentration of 1% to 3%.

[0125] Table 9 Physiological and biochemical characteristics of strain WZSF-1

[0126]

[0127] Note: “+” means positive; “-” means negative

[0128] 3.16S rRNA sequencing and phylogenetic analysis

[0129] 1) DNA extraction of strains

[0130] DNA extraction of strains was carried out according to the method of Yang Wenbo's "Microbiology Experiment". The main steps are as follows:

[0131] (1) WZSF-1 was inoculated on Gao's No. 1 plate and cultured in a constant temperature incubator at 28°C for 7 days. It was then inoculated into YE liquid culture medium and cultured at 28°C for 3 days. The bacterial liquid was collected in a 2 mL centrifuge tube and centrifuged at 10,000 rpm for 5 minutes to collect the bacterial precipitate. Steel beads were added and quickly frozen with liquid nitrogen and then ground using a grinder.

[0132] (2) Add 2× CTAB to the ground cells and incubate in a 65°C water bath for 1 h;

[0133] (3) Add an equal volume of chloroform / isoamyl alcohol (24:1), gently invert to mix, and centrifuge at 12,000 rpm for 5 minutes, retaining the supernatant;

[0134] (4) Add 0.6 volumes of isopropanol to precipitate the DNA, centrifuge at 12,000 rpm for 1 min, and retain the precipitate;

[0135] (5) Wash twice with 70% ethanol, dissolve in TE buffer, and store at -20°C for later use.

[0136] 2) 16S rRNA gene amplification

[0137] The 16S rRNA gene region of the strain was amplified using the universal bacterial identification primers 27F (5'-AGAGTTTGATCCTGGCTCAG-3') and 1492R (5'-GGTTACCTTGTTACGACTT-3') in a 50 μL reaction. The PCR amplification program was as follows: 95°C for 5 min, 94°C for 30 s, 58°C for 50 s, 72°C for 1 min, 30 cycles, and 72°C for 10 min. After PCR amplification, 5 μL of the product was electrophoresed on a 1% agarose gel at 150 V, mA for 30 min. The gel was imaged and photographed on a gel imager. PCR products expressing the desired band were sent to Sangon for sequencing. Sequencing results were compared on NCBI, and sequences with high similarity were downloaded. A phylogenetic tree was constructed using the neighbor-joining method in MEGA 7.0.

[0138] The identification results showed that after PCR amplification using the DNA of strain WZSF-1 as a template, an amplified band of about 1400 was obtained by gel electrophoresis. After sequencing, a 1428 bp 16S rRNA sequence was obtained, as shown in Sequence 1. The sequence was subjected to BLAST comparison analysis on NCBI, and the strain sequences with higher homology were selected. The phylogenetic tree was constructed using the neighbor-joining method on MEGA7.0, as shown in Figure 3 As shown, the strain was clustered into a branch with Streptomyces griseorubens strain C1 (PP112627.1), with a bootstrap value of 98% and a sequence similarity of 99.72% after NCBI BLAST comparison.

[0139] In summary, based on the morphological and cultural characteristics, mycelial morphology, and physiological and biochemical characteristics of the strain, it was identified as Streptomyces griseorubens.

[0140] The above-mentioned strain WZSF-1 was deposited in the Guangdong Provincial Microbiological Culture Collection Center (GDMCC, address: 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou) on October 30, 2024, with the deposit number: GDMCC No. 65368, name: streptomyces griseorubens WZSF-1; classification name: streptomyces griseorubens.

[0141] Example 2: Optimization of Fermentation Conditions for Streptomyces griseorubens WZSF-1

[0142] 1. Preparation of Streptomyces griseorubens WZSF-1 Seed Solution

[0143] A single colony cultured on Gao's No. 1 plate was picked and cultured in 100 mL of YE medium at 28°C with shaking for 3 days.

[0144] 2. Fermentation Carbon Source Optimization

[0145] Single-factor experiments were conducted to screen carbon and nitrogen sources for biocontrol bacteria. 2% soluble starch, glucose, mannose, oatmeal, or maltose were used as carbon sources in the fermentation basal medium, and 1% KNO3 was used as the nitrogen source. The culture medium was inoculated with 1% seed solution of the biocontrol bacteria and cultured at 28°C with shaking for 7 days. The fermentation supernatants were collected and assayed for nematicidal activity. Each treatment was replicated three times.

[0146] 3. Optimization of Fermentation Nitrogen Source

[0147] 1% maltose, soybean powder, beef extract, peptone and potassium nitrate were used as nitrogen sources of the culture medium, and 2% soluble starch was used as carbon source of the culture medium. 1% seed liquid was inoculated and cultured at 28℃ with shaking for 7 days. The nematocidal activity of the fermentation supernatant was determined.

[0148] 4. Fermentation time optimization

[0149] On the basis of screening the optimal carbon and nitrogen sources, different fermentation days were set, and the nematicidal activity of the fermentation supernatant was determined on the 3rd, 5th, 7th, 9th, 11th, 13th and 15th days of fermentation.

[0150] 5. Orthogonal Optimization Experiment

[0151] Based on the results of the above single-factor experiments, four factors were optimized: carbon source, nitrogen source concentration, initial pH, and inoculum size. Three levels were determined for each factor for fermentation. Using orthogonal experiments, the carbon source, nitrogen source concentration, initial pH, and inoculum size of the fermentation medium were determined, using the nematicidal activity of the fermentation supernatant as the evaluation indicator. The orthogonal experimental factors and levels are shown in Table 10.

[0152] Table 10 Orthogonal experimental factors and level design

[0153]

[0154] 6. Nematicidal Activity Assay

[0155] The activity of different fermentation solutions of biocontrol bacteria against the second-instar larvae of the root-knot nematode R. spp. was determined by the immersion method. 900 μL of the fermentation supernatant of actinomycetes was added to a 24-well plate, and then 100 μL of a suspension containing approximately 100 second-instar larvae of the root-knot nematode R. spp. was added and mixed. The mixture was then incubated at room temperature for 24 hours. A blank medium was used as a control, and three replicates were set for each treatment. After treatment, the treated solution was collected into a centrifuge tube and diluted to 8 mL with clean water. The tube was centrifuged at 2000 rpm for 2 minutes, the upper layer was removed, and the solution was diluted to 8 mL with clean water and resuspended for 12 hours. The number of nematode deaths was counted under a dissecting microscope. Nematodes that were immobile and unresponsive to mechanical touch were considered dead. The mortality rate was calculated according to the following formula:

[0156] Nematode mortality rate = number of dead nematodes / total number of tested nematodes × 100%

[0157] Corrected mortality rate = (treatment nematode mortality rate - control nematode mortality rate) / (1 - control nematode mortality rate) × 100%

[0158] 7. Data Analysis

[0159] This experiment used the analysis software IBM SPSS Statistics 27, the variance analysis was performed using Duncan analysis, the data were sorted using WPS software, and the graphs were drawn using Graphpad prism 8.0.

[0160] 8. Results

[0161] 1) Carbon source optimization

[0162] Oatmeal, maltose, soluble starch, glucose and glycerol were used to replace the carbon source in the culture medium, respectively. The results are shown in Table 11. The nematicidal activity of the fermentation supernatant stock solution of each treatment was 78.94%, 88.29%, 91.92%, 78.53% and 86.94%, respectively. Among them, the nematicidal activity was the highest when soluble starch was used as the carbon source, with the nematicidal activity of the stock solution and the 2-fold diluted solution being 91.92% and 84.68%, respectively, which were significantly higher than the nematicidal activity when glucose was used as the carbon source, but not significantly higher than that of the other treatments.

[0163] Table 11 Effects of different carbon sources on the nematocidal activity of WZSF-1 fermentation supernatant

[0164]

[0165]

[0166] Note: Different lowercase letters in the table indicate significant differences in nematocidal activity among different carbon sources (P<0.05)

[0167] 2) Nitrogen source optimization

[0168] The nematicidal activity of the fermentation supernatant of the strain was determined using beef extract, peptone, soybean powder, yeast powder and potassium nitrate as the nitrogen source of the culture medium. The results are shown in Table 12. The average corrected mortality rates of the fermentation supernatant stock solution were 85.83±2.90%, 87.24±5.62%, 94.04±2.71%, 86.13±6.66% and 86.24±3.34%, respectively. The nematicidal activity decreased after being diluted 2-fold, reaching 78.97±3. 43%, 82.85±2.31%, 86.01±5.85%, 78.16±5.78%, and 82.85±3.47%; among them, the nematicidal activity was the greatest when soybean powder was used as the nitrogen source. The nematicidal activities of the original solution and the 2-fold diluted solution were 94.04±2.71% and 86.01±5.85%, respectively. There was no significant difference in the nematicidal activity between the original solution and other nitrogen sources. The nematicidal activity after dilution was significantly higher than that of yeast powder, but there was no significant difference with other nitrogen sources.

[0169] Table 12 Effects of different nitrogen sources on the nematocidal activity of WZSF-1 fermentation supernatant

[0170]

[0171] Note: Different lowercase letters in the table indicate significant differences in nematocidal activity among different nitrogen sources (P<0.05)

[0172] 3) Fermentation time optimization

[0173] The nematicidal activity of the supernatants of the strains was measured at different fermentation times, and the results are shown in Table 13. The nematicidal activity of the fermentation supernatants increased with fermentation time. On the third day, the average corrected mortality rate of the stock solution was 48.71±2.16%, while the activity after a two-fold dilution was only 13.92±0.43%. On the fifth day, the activity exceeded 70%. From the seventh to the fifteenth day, the average corrected mortality rate of the fermentation supernatants was above 80%, indicating that the nematicidal activity tended to be stable, with no significant differences among the treatments.

[0174] Table 13 Effects of different fermentation times on nematocidal activity

[0175]

[0176]

[0177] Note: Different lowercase letters in the table indicate that the nematocidal activity of the fermentation supernatant of the strain at different fermentation days is significant (P<0.05)

[0178] 4) Orthogonal optimization

[0179] Table 14 shows the orthogonal optimization results, using the mortality of second-instar larvae of the root-knot nematode (R. elegans). The order of priority for the factors affecting the nematicidal activity of WZSF-1 was A (carbon source) = B (nitrogen source) > C (initial pH) = D (inoculum size). The optimal combination was A1, B3, C2, and D2. The optimal carbon source concentration, nitrogen source concentration, initial pH, and inoculum size were 1%, 2%, pH 7.0, and 5%, respectively. For example, 1%-3% soluble starch, 1.5% soybean flour, an initial pH of 7, and an inoculum size of 5% increased the nematicidal activity of WZSF-1 from 81.16% to 94.76% after 7 days of shake-flask fermentation.

[0180] Table 14 Effect of orthogonal optimization of fermentation medium on nematocidal activity

[0181]

[0182]

[0183] Example 3: Identification of the insecticidal activity of Streptomyces griseorubens WZSF-1 against southern root-knot nematodes and elephant-ear bean root-knot nematodes

[0184] 1. Obtaining the second-instar larvae of the root-knot nematode

[0185] The roots of peppers that have been colonized by root-knot nematodes are pulled out, the surface soil is washed with clean water, and the root knots are cut into small pieces with scissors; the cut root knots are immersed in clean water, and clean water and sodium hypochlorite solution are added in a volume ratio of 200:1.5, and stirred with a meat grinder for 2-4 times, and then collected in a container and shaken for 5-8 minutes to soften the roots; 100-mesh, 300-mesh, and 500-mesh sieves are stacked from top to bottom, and the softened roots are poured into them. The roots are rinsed with clean water and rubbed by hand to break the root-knot nematode eggs; the 500-mesh sieve is repeatedly rinsed with running water to remove residual sodium hypochlorite until there is no irritating smell, and the root-knot nematode eggs on the 500-mesh sieve are collected and placed on a double layer of lens paper, immersed in clean water and incubated at room temperature. The hatching status can be observed daily under a dissecting microscope; finally, the hatched second-instar larvae are collected and prepared into an adult suspension for later use.

[0186] 2. Obtaining the second-instar larvae of southern root-knot nematode

[0187] The method for obtaining southern root-knot nematodes refers to the method for obtaining elephant ear bean root-knot nematodes in step 1.

[0188] 3. Preparation of strain fermentation broth

[0189] 1) Activation of Streptomyces griseorubens WZSF-1 strain

[0190] The preserved actinomycetes were inoculated on Gao's No. 1 plate and cultured at 28°C for 5 to 7 days.

[0191] 2) Preparation of seed solution of Streptomyces griseorubens WZSF-1

[0192] A single colony was picked from the activated actinomycete plate with a sterile toothpick and cultured in 100 mL of optimized culture medium at 28°C with shaking for 3 days.

[0193] 3) Fermentation broth preparation

[0194] The strain seed liquid was inoculated into the optimized liquid culture medium at a 1% inoculum volume and cultured at 28°C for 7 days with shaking. The fermentation broth of each strain was centrifuged at 10,000 rpm for 10 minutes to obtain the fermentation supernatant of the strain, which was stored at 4°C for later use.

[0195] 4. Determination of nematicidal activity

[0196] The activity of the biocontrol bacteria Streptomyces griseorubens WZSF-1 against southern root-knot nematodes and elephant-ear bean root-knot nematodes was determined using the immersion method. 900 μL of the fermentation supernatant or dilution was added to a 24-well plate. 100 μL of a suspension containing approximately 100 southern root-knot nematodes or elephant-ear bean root-knot nematodes was then added and mixed. The mixture was then incubated at room temperature for 24 hours. A blank medium was used as a control, and three replicates were set up for each treatment. After treatment, the treated solution was collected into a centrifuge tube and diluted to 8 mL with water. The tube was centrifuged at 2000 rpm for 2 minutes. The supernatant was removed, diluted to 8 mL with water, and resuspended for 12 hours. Nematodes were counted and counted under a dissecting microscope. Nematodes that were immobile and unresponsive to mechanical touch were considered dead. Mortality was calculated according to the following formula:

[0197] Nematode mortality rate = number of dead nematodes / total number of tested nematodes × 100%

[0198] Corrected mortality rate = (treatment nematode mortality rate - control nematode mortality rate) / (1 - control nematode mortality rate) × 100%

[0199] 5 Results

[0200] The results are shown in Table 15. As can be seen from the table, the fermentation supernatants of the WZSF-1 strain at different dilution ratios all showed good nematicidal activity against the second-instar larvae of the southern root-knot nematode and there was no significant difference between them. The highest dilution ratio was 90.22%, which was slightly lower than the nematicidal activity of the second-instar larvae of the ear bean root-knot nematode at 48 hours.

[0201] Table 15 Nematicidal activity of fermentation supernatants of WZSF-1 strain at different dilutions against second-instar larvae of R. aurantii and R. incognita

[0202]

[0203] Example 4: Potted control efficacy test of Streptomyces griseorubens WZSF-1 against southern root-knot nematodes and elephant-ear bean root-knot nematodes

[0204] 1. Potted experiment design

[0205] Pepper seedlings of uniform size at the 3-4 leaf stage were transplanted into pots and treated after a week of acclimatization. This experiment included four treatments: 1) Control (CK): Inoculated with 2,000 nematodes; 2) Treatment 1 (AW): Inoculated with 2,000 nematodes and treated with a 1,000-fold dilution of 5% Avermectin emulsifiable concentrate; 3) Treatment 2 (F1): Inoculated with 2,000 nematodes and treated with a fermentation supernatant of Streptomyces griseorubens WZSF-1; and 4) Treatment 3 (F2): Inoculated with 2,000 nematodes and treated with a 5-fold dilution of the fermentation supernatant of Streptomyces griseorubens WZSF-1. Each treatment was irrigated with 100 mL of water per pot, with six pepper seedlings per treatment replicated.

[0206] 2. Statistics on prevention and control effects

[0207] 1) Determination of protective efficacy

[0208] On the 60th day after treatment, the pepper seedlings were pulled out, the soil around the roots was washed clean with running water, and photos were taken. The root knot index of peppers was graded according to the following grading criteria, and the control effect was calculated.

[0209] Level 0: No Root Knot

[0210] Level 1: There are a small number of root knots, accounting for 1%-25% of the whole root system;

[0211] Level 2: The number of root nodes is moderate, accounting for 26%-50% of the total root system;

[0212] Level 3: The number of root nodes is large, accounting for 51%-75% of the whole root system;

[0213] Level 4: There are a large number of root nodes, accounting for 76%-100% of the entire root system.

[0214] Root knot index = ∑[(level × number of diseased plants in this level)] / (total number of plants surveyed × highest level) × 100%

[0215] Control effect = (root knot index of control group - root knot index of treatment group) / root knot index of control group × 100%

[0216] 2) Physiological index measurement

[0217] The growth indices of pepper were determined; chlorophyll content, plant height, stem diameter, aboveground fresh weight and root weight of pepper were measured 60 days after treatment.

[0218] 3. Determination of pepper defense enzyme activity

[0219] Extraction of crude enzyme solution: Chop the treated leaves and mix them evenly. Weigh 0.1g of leaves and place them in a mortar. Add 2mL of the extract in the kit. Grind thoroughly and transfer to a 1mL centrifuge tube. Centrifuge at 10,000g / min for 10min. Take the supernatant for use and store in a 4℃ refrigerator for short-term storage.

[0220] The contents of phenylalanine ammonia lyase (PAL), catalase (CAT), polyphenol oxidase (PPO), and malondialdehyde (MDA) in pepper leaves were determined according to the reagent instructions.

[0221] 4. Data analysis software

[0222] This experiment used the analysis software IBM SPSS Statistics 27, the variance analysis was performed using Duncan analysis, the data were sorted using WPS software, and the graphs were drawn using Graphpad prism 8.0.

[0223] 5 Results

[0224] (1) Effect of biocontrol bacteria on pepper root-knot nematodes

[0225] The root knot index of pepper after 60 days of treatment is as follows Figure 4 As shown in the figure, the root knot indexes of CK, avermectin (AW), WZSF-1 stock solution and 5-fold dilution of WZSF-1 were 100%, 18.75%, 50.00% and 56.25% respectively, and the control efficacy on pepper was 81.25%, 50.00% and 43.75% respectively. The control efficacy of the strain decreased after dilution.

[0226] (2) Impact on pepper growth

[0227] The results of each treatment on the growth of pepper are as follows Figure 5 、 Figure 6 and Figure 7 The chlorophyll content, plant height, stem diameter, aboveground fresh weight and root weight of the AW and WZSF-1 treatment groups were all promoted.

[0228] After applying the original solution and diluted solution of the fermentation supernatant of the biocontrol bacteria, the chlorophyll content increased extremely significantly and significantly compared with CK, the plant height and stem diameter increased significantly, the fresh weight of the aboveground part increased extremely significantly, and the root weight increased significantly and extremely significantly after applying the original solution and diluted solution. The fermentation supernatant of WZSF-1 has a significant promoting effect on pepper growth.

[0229] (3) Effects on defense enzyme activity and malondialdehyde content

[0230] a. Phenylalanine ammonia lyase (PAL) activity

[0231] from Figure 8It can be seen that after applying the fermentation supernatant of biocontrol bacteria, the PAL enzyme activity of pepper leaves treated with AW and WZSF-1 decreased, but the difference with CK was not significant. After 5-fold dilution, the enzyme activity content of each treatment was lower than that of the original solution.

[0232] b. Catalase (CAT) activity

[0233] The results of the effects of each treatment on the CAT enzyme activity in pepper leaves are as follows Figure 9 After applying the fermentation supernatant of the biocontrol bacteria, CAT enzyme activity increased in peppers across all treatments, and CAT activity increased with increasing dilution. Compared with CK, CAT enzyme activity in the WZSF-1 dilution and AW treatments increased significantly. The 5-fold dilution significantly increased CAT enzyme activity compared to the original solution. The AW treatment had the highest enzyme activity, at 1075.40 U / g, followed by the F2 treatment, at 1072.85 U / g.

[0234] c. Polyphenol oxidase (PPO) activity

[0235] The results of PPO enzyme activity determination in pepper leaves are as follows Figure 10 Compared with CK, the enzyme activities of each treatment increased. The AW and dilution treatments were significantly higher than CK, with enzyme activities of 162.00 U / g and 146.40 U / g, respectively. The enzyme activity of the stock solution treatment had no significant difference from that of CK.

[0236] d. Malondialdehyde (MDA) content

[0237] The results of MDA content determination in pepper leaves are as follows Figure 11 The MDA content in pepper leaves increased after AW treatment, and decreased after application of WZSF-1 stock solution and its 5-fold dilution, but there was no significant difference between each treatment and CK.

[0238] in conclusion

[0239] 1. The immersion method was used to screen out the actinomycete strain WZSF-1 with high and stable nematicidal activity against the second-instar larvae of the ear bean root-knot nematode and the southern root-knot nematode. Through culture characteristics, scanning electron microscopy characteristics, physiological and biochemical characteristics, and molecular biological identification, and by constructing a phylogenetic tree using the neighbor-joining method, WZSF-1 was determined to be Streptomyces griseorubens.

[0240] 2. The fermentation conditions of WZSF-1 were investigated by single-factor and orthogonal experiments. The results showed that 1%-3% soluble starch, 1.5% soybean powder, an initial pH value of 7, and an inoculation amount of 5% increased its nematicidal activity from 81.16% to 94.76 after 7 days of shake flask fermentation.

[0241] 3. The results of the potted test showed that WZSF-1 had a good biocontrol effect on pepper root-knot nematode disease, with the highest control effect of 50.00%. After the application of actinomycetes, the chlorophyll, stem diameter, and plant height of the pepper plants were higher than those of the control group; after the WZSF-1 treatment, the PAL enzyme activity and MDA content decreased, and the CAT and PPO defense enzyme activities increased.

Claims

1. An actinomycete that kills root-knot nematodes, characterized by: The actinomycete is named Streptomyces griseorubens WZSF-1, classified as Streptomyces griseorubens, and its deposit number in Guangdong Provincial Microbiological Culture Collection Center is GDMCC No. 65368.

2. Use of the actinomycete according to claim 1 in preventing and controlling root-knot nematodes.

3. The use according to claim 2, characterized in that The root-knot nematodes are root-knot nematodes of southern origin and / or root-knot nematodes of elephant ear bean origin.

4. A biological product containing the actinomycete according to claim 1, wherein the biological product is used for controlling root-knot nematodes.

5. The biological product according to claim 4, characterized in that The root-knot nematodes are root-knot nematodes of southern origin and / or root-knot nematodes of elephant ear bean origin.

6. The method for culturing the actinomycetes according to claim 1, wherein the actinomycetes are inoculated into a culture medium containing 1%-3% soluble starch, 1.5% soybean powder, and an initial pH value of 7, wherein the "%" represents the percentage by mass.

7. The method for culturing actinomycetes according to claim 6, wherein The inoculation volume percentage of the inoculation is 5%.

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