A Streptomyces aureus and its application in controlling damping-off of cucumber

Abstract

The present invention discloses an actinomycete with antagonistic effect against the pathogen of cucumber damping-off and its application. The actinomycete is Streptomyces aureusensis Q1C-44, which has antagonistic effect against Rhizoctonia solani of cucumber, can effectively inhibit the hyphal elongation of the pathogenic fungus, and can be used to control damping-off caused by Rhizoctonia solani of cucumber.

Description

Technical Field

[0001] The present invention relates to a strain of Streptomyces aureusensis with antagonistic effect against Rhizoctonia solani of cucumbers and its application in controlling cucumber damping-off disease. Background Art

[0002] With the development of agriculture, the types of planted crops are increasing continuously, and various pest and disease problems are becoming increasingly prominent. The development of green agriculture is the development direction of agriculture in the new era. Currently, the contradiction among resources, land and environment is tense, and the healthy development of agriculture has attracted more and more attention. Although the extensive use of chemical pesticides can inhibit pests and diseases to a certain extent, it will also cause great harm to the environment. Using biological control of pests and diseases is undoubtedly an effective, safe and environmentally friendly method. The biological control method actively responds to China's sustainable development strategy and plays an important role in the sustainable development of agriculture and the protection of land environment.

[0003] As an economic crop, cucumbers are widely popular across the country. Cucumber damping-off disease is a common disease of cucumbers, and the pathogen is Rhizoctonia solani, a fungus in the Deuteromycotina. The pathogen overwinters in the soil or diseased tissues in the form of mycelium or sclerotia, with strong saprophytic ability. Generally, it can survive in the soil for 2 - 3 years and can be spread through rainwater, running water, farm tools and contaminated compost. Cucumber damping-off disease at the seedling stage is one of the main soil-borne diseases of vegetables and is the main limiting factor affecting the quality and survival rate of seedlings. With the increase of continuous cropping years, the accumulation of pathogens leads to increasing harm year by year. It occurs in varying degrees across the country, especially in the middle and late stages of seedling raising, often causing a large number of dead seedlings or even the destruction of the seedbed. Cucumber damping-off disease can also harm the seedlings of tomatoes, eggplants, peppers, beans and other crops in addition to cucumbers. Cucumber damping-off disease deeply troubles cucumber growers and also has an adverse impact on people's vegetable baskets.

[0004] Nowadays, among biological pesticides, microbial pesticides mainly include living microbial pesticides and metabolite pesticides, and have attracted attention due to their good environmental compatibility. Microbial fertilizers have obvious control effects on plant diseases such as root rot, leaf mold, and damping-off caused by fungi and molds. Microbial fertilizers use bacteria to control bacteria, that is, adopt biological control methods to prevent and control plant diseases and insect pests, which can reduce the use of chemical pesticides and ultimately eliminate the use of chemical pesticides, so as to achieve the goal of producing green agricultural products and organic foods. The ocean is rich in biological resources, and there are abundant actinomycetes in marine sediments, containing more rare actinomycete groups than the terrestrial environment. Marine actinomycetes often produce a variety of natural active substances, which have obvious inhibitory effects on bacteria and fungi. In this invention, actinomycetes in the ocean are used as the research object. Through experiments such as the isolation and screening of actinomycetes and the control of diseases, they are made into microbial preparations and applied to green agricultural production, showing broad development prospects. Summary of the Invention

[0005] The purpose of the present invention is to provide an actinomycete with antagonistic effect against Rhizoctonia solani of cucumber and its application in controlling cucumber damping-off.

[0006] The actinomycete with antagonistic effect against Rhizoctonia solani of cucumber provided by the present invention is Streptomyces aureusensis Q1C-44CGMCC No. 24891.

[0007] The Streptomyces aureusensis Q1C-44 has been deposited at the General Microbiological Center of the China Committee for Culture Collection of Microorganisms (abbreviation: CGMCC, address: No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, China) on May 12, 2022, and the deposit number is No. 24891.

[0008] The Streptomyces aureusensis Q1C-44 is isolated from the sediments of Qilihai Lagoon in Beidaihe and is a Gram-positive bacterium. The aerial mycelium of this actinomycete is light yellow on PSA medium, the substrate mycelium is golden yellow, there is no soluble pigment, the spore hypha is tightly spiral, the spores are arranged in chains and are round. It can produce weak lipase and protease activities.

[0009] The biological bactericide or microbial fertilizer with Streptomyces aureusensis Q1C-44No. 24891 as the active ingredient also belongs to the protection scope of the present invention.

[0010] The actinomycete with antagonistic effect against Rhizoctonia solani of the present invention is Streptomyces aureusensis Q1C-44, which is isolated from the sediment of Qilihai Lagoon in Beidaihe. The plate confrontation experiment shows that this actinomycete has a good antagonistic effect against Rhizoctonia solani causing cucumber damping-off. In the confrontation culture, the growth inhibition rate of this actinomycete Q1C-44 against Rhizoctonia solani of cucumber is 66.7%. It can significantly make the hyphae length at the edge of the pathogenic bacterial colony significantly shorter, the hyphae thinner and more tortuous, and the color turns yellow. The pot experiment shows that this actinomycete has a good control effect on the damping-off caused by Rhizoctonia solani of cucumber. Streptomyces aureusensis Q1C-44 in the present invention is a strain with good biocontrol application prospect, and the acquisition of this strain is expected to provide an environmental-friendly, simple and effective way for the control of cucumber damping-off. BRIEF DESCRIPTION OF THE DRAWINGS

[0011] Figure 1 It is the physiological characteristics of the inhibition of Rhizoctonia solani of cucumber by actinomycete Q1C-44.

[0012] Figure 2 It is the zonal observation of the physiological characteristics of the inhibition of Rhizoctonia solani of cucumber by actinomycete Q1C-44 (A: Observation of the inhibition zone; B: Observation of the Rhizoctonia solani colony in the inhibition zone; C: Observation of the non-inhibition zone; D: Observation of the Rhizoctonia solani colony in the non-inhibition zone).

[0013] Figure 3 It is the control effect of actinomycete Q1C-44 as a biological agent on cucumber damping-off (A: Control group: soil containing Rhizoctonia solani, Experimental group: soil containing Rhizoctonia solani and actinomycete, Blank group: ordinary soil; B: Cucumber seedlings in the control group; C: Cucumber seedlings in the experimental group; D: Cucumber seedlings in the blank group).

[0014] Figure 4 It is the effect of actinomycete Q1C-44 as a biological agent on the stem length of cucumber. DETAILED DESCRIPTION OF THE INVENTION

[0015] The methods in the following examples are all conventional methods unless otherwise specified.

[0016] The percentage contents in the following examples are all mass percentage contents unless otherwise specified.

[0017] Example 1. Screening of Actinomycetes Antagonistic to Rhizoctonia solani

[0018] I. Isolation and Preservation of Actinomycetes

[0019] Collect 5-10 cm samples of sediment from the surface of Qilihai Lagoon in Beidaihe, weigh 10 g of sediment, put it into a triangular flask containing 90 ml of sterile water, and shake it vigorously for 30 min to obtain a dilution of 10. -1 Take 1 ml of the above sediment bacterial suspension, add it to a test tube containing 9 ml of sterile water, vortex and mix well to obtain a dilution of 10 -2 10-fold serial dilutions were made to 10 -4 . Take 10 respectively -1 Up to 10 -4 20 μl of the bacterial suspension was evenly spread on a plate with Gao's medium No. 1 (soluble starch 20 g / L, KNO 3 1g / L, NaCl 0.5g / L, K 2 HPO 4 ·3H 2 O 0.5g / L, MgSO 4 7H 2 O0.5g / L, FeSO 4 7H 2 O0.5g / L, agar 15g / L, pH 7.2-7.4; after the medium is sterilized, K is added to a final concentration of 50mg / L 2 Cr 2 O 7 , then invert the plate). Spread the plate evenly, and after the bacterial suspension on the surface dries, seal the plate with a sealing film, and then invert and culture it in a constant temperature incubator at 30°C for 7 days. Until colonies appear, pick a single colony and further streak culture on a PSA plate (potato 200g / L, sucrose 20g / L, agar 20g / L), and culture it at 30°C for 7 days to obtain a pure culture of bacteria. Scrape off the actinomycete moss on the PSA culture medium plate, put it into a sterile 2ml centrifuge tube, and then add CM liquid culture medium (casein amino acids 7.5g / L, yeast powder 10g / L, sodium citrate 3g / L, MgSO 4 7H 2 O 20g / L, KCl 2g / L, FeSO 4 7H 2 O 0.05g / L, NaCl 50g / L), vortexed and then stored in a -70°C refrigerator. Finally, 33 pure cultured actinomycetes were isolated.

[0020] 2. Screening of actinomycetes against Rhizoctonia solani

[0021] The screening of actinomycetes against Rhizoctonia solani was carried out by confrontation culture. A small amount of mycelium of the activated Rhizoctonia solani was inoculated in the center of the PSA medium plate.6 A bacterial suspension of [[[spore concentration]]] spores / ml was prepared, and then 2 μL was pipetted and inoculated at both ends 3 cm away from the center of the plate, with the two ends without inoculating actinomycetes as the control. The edges of the inoculated plates were sealed with sealing film and then cultured at 30 °C. When the leading edge hyphae of [[[Rhizoctonia solani]]] inoculated in the center of the plate grew 3 cm on the side without actinomycetes, the colony radius of the pathogenic fungus on the side inoculated with actinomycetes was measured, and according to the formula: Inhibition rate = [(3 cm - actual growth radius of [[[Rhizoctonia solani]]] colony) / 3 cm]×100%, the inhibition rates of various actinomycetes against the pathogenic fungus were calculated and compared.

[0022] Thirty-three strains of actinomycetes were co-cultured with [[[Rhizoctonia solani]]] respectively. Eleven strains of actinomycetes could antagonize [[[Rhizoctonia solani]]], and their inhibition rates were between 20.0% and 66.7% (Table 1). Among them, Q1C-44 had the highest inhibition rate against [[[Rhizoctonia solani]]], reaching 66.7%. The antagonistic effect of this actinomycete against [[[Rhizoctonia solani]]] of cucumber is as Figure 1 shown.

[0023] Table 1. Results of confrontation culture of 33 strains of actinomycetes and [[[Rhizoctonia solani]]] of cucumber

[0024]

[0025]

[0026] Note: "+++" indicates strong inhibition ability, inhibition rate > 60%; "++" indicates medium inhibition ability, inhibition rate is 30% - 60%; "+" indicates weak inhibition ability, inhibition rate ≤ 30%; "-" indicates no inhibition.

[0027] Example 2. Physiological and biochemical characteristics and species identification of strain Q1C-44

[0028] I. Physiological and biochemical characteristics of the strain

[0029] Method for observing colony color and morphology. The preserved strain Q1C-44 was taken and streaked continuously on Gause's No. 1 plate and PSA plate by the plate streaking method. The streaked plates were inverted and cultured in an incubator at 30 °C until single colonies appeared. Method for detecting enzyme production by the strain. Lipase detection medium: beef extract 3 g / L, peptone 10 g / L, tributyrin 6 ml / L, NaCl 10 g / L, agar 20 g / L. Cellulose detection medium: carboxymethyl cellulose 5 g / L, NaNO 3 1 g / L, K 2 HPO 4 2 g / L, KCl 1 g / L, MgSO 4 .7H 2 It should be noted that some parts in the original text seem to have incomplete chemical formula expressions or unclear content, which are translated as accurately as possible according to the existing information. For example, the incomplete chemical formula parts are left as they are in the translation. You may need to check and supplement the complete information for a more accurate translation in a real situation.O 0.5 g / L, yeast powder 2 g / L, glucose 1 g / L, NaCl 10 g / L, agar 20 g / L. Protease detection medium: skim milk powder 10 g / L, yeast powder 2 g / L, NaCl 10 g / L, agar 20 g / L. Amylase detection medium: starch 5 g / L, yeast powder 2 g / L, (NH 4 ) 2 SO 4 1.4 g / L, K 2 KPO 4 2 g / L, MgSO 4 ·7H 2 O 0.2 g / L, NaCl 10 g / L, agar 20 g / L. The activated strain Q1C-44 was inoculated separately in the center of the lipase detection, cellulase detection, protease detection, and amylase detection plates, and cultured in an inverted position in an incubator at 30 °C for 5 d. If visible transparent circles are formed on the lipase detection medium and protease detection medium by this strain, it indicates the production of lipase and protease; the cellulase detection plate was rinsed with 0.1% Congo red. If a transparent circle appears around the colony, it indicates that the strain produces cellulase; the amylase detection plate was rinsed with 0.3% iodine solution. If a transparent circle appears around the colony, it indicates that the strain produces amylase.

[0030] The physiological and biochemical test results are as follows:

[0031] On the PSA medium, the aerial mycelium of this strain is light yellow, the substrate mycelium is golden yellow, there is no soluble pigment, the spore hyphae are tightly spiral-shaped, and the spores are arranged in chains and are round.

[0032] This strain can produce lipase and protease with relatively low activity; the results are shown in Table 1.

[0033] Table 1. Enzyme production detection of Q1C-44

[0034] Enzyme type Enzyme production Diameter of clear zone (cm) Lipase + 1.30±0.01 Cellulase - 0 Protease + 0.65±0.01 Amylase - 0

[0035] Note: "+" indicates production; "-" indicates non-production; the experiment was set with three replicates.

[0036] II. 16S rRNA gene sequence and identification of strain Q1C-44

[0037] The genomic DNA of Actinomyces Q1C-44 was extracted. Using this DNA as a template, PCR amplification was performed with the universal 16S rRNA primers for bacteria (27f: 5’-GTTTGATCCTGGCTCAG-3’, 1492r: 5’-CTACGGCTACCTTGTT-3’). After the PCR amplification products were subjected to agarose gel electrophoresis and purified by gel cutting and recovery, sequencing was performed at both ends with the 27f and 1492r primers respectively. After sequence splicing, a sequence with a total length of 1370 bp was obtained, and the sequence is shown as Sequence 1 in the sequence listing. This sequence was compared and analyzed in the NCBI database (https: / / www.ncbi.nlm.nih.gov / ), and the results showed that the 16S rRNA gene sequence of this strain had the highest similarity with the 16S rRNA gene sequence of Streptomyces aureusensis (AGR0001) in the database, and the similarity between the two was 99.93%.

[0038] Based on the 16S rRNA sequence characteristics of this actinomycete, it was named Streptomyces aureusensis Q1C-44. The Streptomyces aureusensis Q1C-44 was deposited on May 12, 2022 at the General Microbiology Center of the China Committee for Culture Collection of Microorganisms (abbreviated as CGMCC, address: No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, China), and the deposit number is No. 24891.

[0039] Example 3. Inhibitory Physiological Characteristics of Streptomyces aureusensis Q1C-44 CGMCC No. 24891 against Rhizoctonia solani of Cucumber

[0040] I. Physiological Characteristics of the Inhibition of Q1C-44 against Rhizoctonia solani of Cucumber in Confrontation Culture

[0041] According to the method described in the second step of Example 1, Streptomyces aureusensis Q1C-44 and Rhizoctonia solani of cucumber were subjected to confrontation culture, and then cultured at 30°C. When the leading-edge hyphae of the pathogenic fungus grew to 3 cm on the side without inoculating the actinomycete, the inhibition rate of Actinomyces Q1C-44 against the pathogenic fungus of cucumber was calculated, and the physiological characteristics of the inhibition were observed. In order to more clearly observe the inhibitory effect of Q1C-44 on the pathogenic fungus, the method of three-point plate confrontation culture was adopted, and the results are shown in Figure 1 , from which it can be seen that Q1C-44 has an obvious inhibitory effect on Rhizoctonia solani (Table 2, Figure 2 ).

[0042] Table 2. Physiological characteristics and inhibition rate of the inhibition of Rhizoctonia solani by Q1C-44 colonies against cucumber

[0043]

[0044] As can be seen from the above results, during the confrontation culture process, the actinomycete Q1C-44 showed obvious inhibitory physiological characteristics against Rhizoctonia solani of cucumber, and its inhibition rate was 66.7%.

[0045] Example 4. Control effect of Streptomyces aureusensis Q1C-44 CGMCC No. 24891 on cucumber damping-off

[0046] I. Preparation of soil containing pathogenic fungi

[0047] Propagation of pathogenic fungi. Pick the activated mycelia of Rhizoctonia solani of cucumber and inoculate them in the center of a PSA medium plate, and place them in an incubator at 30 °C for cultivation; after 5 days of cultivation, reserve them for use.

[0048] Preparation of garden soil. Take a certain amount of soil from the garden of China University of Geosciences (Beijing), grind and crush it, sieve it to make the soil texture uniform, and then put it into a triangular flask for high-pressure steam sterilization for 1 h, and reserve it after cooling.

[0049] Preparation of soil. Three treatments, namely a blank group, a control group and an experimental group, are set up for the preparation of soil. Put 100 g of sterilized soil into a plastic cup as the blank group (the plastic cup is pre-drilled with 5 small holes of equivalent diameter at the bottom); mix Rhizoctonia solani of cucumber and sterilized soil at a ratio of 1:100, and transfer the mixed bacteria and soil into the plastic cup as the control group; the soil in the experimental group is treated in the same way as the control group. Each treatment has three replicates.

[0050] II. Pot experiment

[0051] Preparation of cucumber seeds. Soak the cucumber seeds overnight, and then germinate them. After the seeds grow about 3 cm long buds, select cucumber seedlings with similar germination degree and size, and reserve them for use.

[0052] Sowing and management. Sow the germinated cucumber seedlings into the plastic cups of the blank group, the control group and the experimental group respectively, 5 plants per cup. Among them, the cucumber seedlings in the experimental group are first dipped in about 0.1 g of Streptomyces aureus with their roots and then planted. Each treatment is cultured at room temperature, and watered by irrigation from the bottom, once every 2 days, 50 ml each time.

[0053] Results and statistics. When the cucumbers grow to 7 days, take out the soil from the plastic cups and wash off the soil on the cucumber roots ( Figure 3), a comprehensive evaluation was conducted on each cucumber in each treatment, measuring the root length and plant height of the cucumbers. The results are shown in Figure 4 .

[0054] As can be seen from the above results, the cucumbers in the blank group grew well and luxuriantly because they grew in sterilized soil; while the cucumbers in the control group showed obvious disease symptoms as they grew in diseased soil containing *Rhizoctonia solani* of cucumbers. Compared with the blank group, the stems were thin and the growth was slow. Finally, the cucumber seedlings toppled over. Upon examination of their roots, it was found that the roots of the cucumber seedlings rotted, and the remaining roots were brown or dark brown. Eventually, the cucumber seedlings died due to root rot ( Figure 3 ). The cucumbers in the experimental group had their roots dipped in the spores of *Actinomyces* Q1C-44. The metabolites produced by the *Actinomyces* inhibited the growth of *Rhizoctonia solani* of cucumbers to a certain extent, resulting in the cucumber roots being less damaged or protected from *Rhizoctonia solani*. Compared with the control group, obvious roots could be observed, and the stem length also increased significantly; compared with the blank group, its roots were similar to those of the blank group, and its stem length was slightly lower than that of the blank group ( Figure 3 and Figure 4 ). The overall growth status was similar to that of the blank group. The above experimental results indicate that pre-applying *Actinomyces* Q1C-44 to the roots of cucumber seedlings can effectively control cucumber damping-off caused by *Rhizoctonia solani* to a certain extent. Its biological agent can be used to control cucumber damping-off and achieve good control effects.

[0055] The above description is illustrative rather than restrictive for the present invention. Those of ordinary skill in the art understand that many modifications, variations or equivalents can be made without departing from the spirit and scope defined by the appended claims, and all of them will fall within the protection scope of the present invention.

Claims

1. A Streptomyces aureus strain ( Streptomyces aureusensis ) Q1C-44, with the preservation number of CGMCC No. 24891.

2. Use of Streptomyces aureus ( Streptomyces aureusensis ) Q1C-44 in preventing and controlling plant diseases; the plant disease is damping-off caused by Rhizoctonia solani cucumeris.

3. Use of Streptomyces aureus ( Streptomyces aureusensis ) Q1C-44 in preparing a biocontrol agent or microbial fertilizer against Rhizoctonia solani Kühn of cucumber.

4. A biocontrol agent for preventing and controlling plant diseases, characterized in that, The active ingredient of the biocontrol agent is Streptomyces aureus ( Streptomyces aureusensis ) Q1C-44 as claimed in claim 1; the plant disease is damping-off caused by Rhizoctonia solani of cucumber.

5. A microbial fertilizer for preventing and controlling plant pathogenic fungi, characterized in that, The active ingredient of the microbial bacterial fertilizer is Streptomyces aureus ( Streptomyces aureusensis ) Q1C-44 as claimed in claim 1; the plant pathogenic fungus is Rhizoctonia solani of cucumber.

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