Herbaspirillum seropedicae strain ZJUCS 1002 and its uses

Abstract

The present invention relates to the field of microorganisms, and particularly relates to a Herbaspirillum seropedicae strain ZJUCS1002 and its control effect on tea tree anthracnose. The present invention discloses a Herbaspirillum seropedicae strain ZJUCS 1002, with the preservation number: CGMCC NO: 24723. The present invention also simultaneously discloses the use of the Herbaspirillum seropedicae strain ZJUCS 1002: inhibiting tea tree anthracnose and controlling tea tree anthracnose.

Description

Technical Field

[0001] The present invention relates to the field of microorganisms, and particularly to a Herbaspirillum seropedicae strain ZJUCS1002 and its control effect on tea tree anthracnose. Background Art

[0002] Tea plant endophytic bacteria refer to all bacteria that can colonize inside tea plants without causing obvious harm to the host during a certain period of their life, and belong to prokaryotes. Common ones include Actinomycetia, Bacilli, and Betaproteobacteria, etc. Tea plant endophytes can be isolated from the roots, stems, leaves, flowers, and fruits of various tea plant varieties. With the in-depth research, endophytes are considered a new type of microbial resource with great research value and broad application prospects. After the pathogen infects the tea tree, the quantity and types of tea plant endophytes have changed significantly. Endophytes can antagonize pathogens by means of nutrient and space competition, producing anti-pathogen secondary metabolites, and inducing the expression of host defense-related genes, thereby reducing the incidence of diseases. In addition, endophytes have the advantage of not causing infection or disease in the host, so endophytes are potential candidate strain sources for biological control applications.

[0003] The currently known uses of Herbaspirillum seropedicae are as follows:

[0004] 1. "Screening of the Rice Endophytic Nitrogen-Fixing Bacterium Herbaspirillum seropedicae DX35 and Its Growth-Promoting Characteristics" informs that DX35 belongs to H. seropedicae and is a highly efficient endophytic nitrogen-fixing bacterium.

[0005] 2. "Herbaspirillum seropedicae promotes maize growth but fails to control the maize leaf anthracnose" informs that SmR1 belongs to H. seropedicae, has the function of promoting maize growth, but cannot inhibit the occurrence of maize anthracnose.

[0006] 3. "Substrate biofortification in combination with foliar sprays of plant growth promoting bacteria and humic substances boosts production of organic tomatoes" informs that BR 11335 belongs to H. seropedicae and can promote the growth of tomato seedlings. Summary of the Invention

[0007] The technical problem to be solved by the present invention is to provide a novel strain of Herbaspirillum seropedicae, Herbaspirillum seropedicae ZJUCS 1002, and its use, which can prevent and control anthracnose of tea trees.

[0008] To solve the above technical problem, the present invention provides a strain of Herbaspirillum seropedicae ZJUCS 1002, with the preservation number: CGMCC NO: 24723.

[0009] The preservation information of strain ZJUCS 1002 is as follows: the preservation name is Herbaspirillum seropedicae, the preservation unit is the General Microbiology Center of the China Committee for Culture Collection of Microorganisms, the preservation address is No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, the preservation date is April 19, 2022, and the preservation number is CGMCC NO: 24723.

[0010] The strain Herbaspirillum seropedicae ZJUCS 1002 provided by the present invention has the following taxonomic status: strain ZJUCS 1002 belongs to the kingdom Bacteria, phylum Proteobacteria, class Betaproteobacteria, order Burkholderiales, family Oxalobacteraceae, and genus Herbaspirillum.

[0011] The present invention also simultaneously provides the use of the above strain: inhibiting and preventing anthracnose of tea trees. That is, inhibiting the mycelial growth of Colletotrichum camelliae, the pathogen causing anthracnose of tea trees, and reducing the infection rate of anthracnose on tea tree leaves. Inhibiting the growth of the pathogen causing anthracnose of tea trees includes inhibiting the mycelial growth of Colletotrichum camelliae.

[0012] The strains involved in the present invention can achieve the following beneficial effects:

[0013] 1. The inhibition rate of mycelial growth against Colletotrichum camelliae reaches 34.44%.

[0014] 2. It effectively reduces the infection rate of anthracnose on tea tree leaves by 31.01%.

[0015] 3. The control effect of Herbaspirillum seropedicae strain on anthracnose of tea tree reaches 54.9%.

[0016] In summary, Herbaspirillum seropedicae of the present invention has the function of controlling anthracnose of tea tree, which is completely different from the nitrogen fixation or plant growth promotion effects of the existing Herbaspirillum seropedicae. Brief Description of the Drawings

[0017] The following further elaborates on the specific implementation manners of the present invention with reference to the drawings.

[0018] Figure 1 It is a result diagram of the plate inhibition experiment of Herbaspirillum seropedicae. Compared with the control CK (left), the strain of Herbaspirillum seropedicae ZJUCS 1002 (right) inhibits the growth of Colletotrichum camelliae on the PDA plate. Among the strains of the same Herbaspirillum genus, there is also a certain effect of inhibiting the growth of Colletotrichum camelliae (middle);

[0019] Figure 2 It is the antibacterial and disease-resistant experiment of Herbaspirillum seropedicae on tea trees. After inoculating the tea tree anthracnose fungus Colletotrichum camellia and the Herbaspirillum seropedicae ZJUCS 1002 of the present invention and the strain of Herbaspirillum huttiense belonging to the same classification, the tea tree anthracnose fungus Colletotrichum camellia is inoculated on its leaves, and at the same time, the inoculation of the Herbaspirillum seropedicae ZJUCS 1002 strain reduces the incidence of anthracnose on tea tree leaves.

[0020] Figure 3Statistical chart of the incidence of antibacterial and disease-resistant experiments of Herbaspirillum huttiense on tea plants. Among them, the incidence of anthracnose on the tea tree leaves in the control group was 56.47%, the incidence of the group inoculated with the Herbaspirillum huttiense strain was 39.16%, and the incidence of the group inoculated with Herbaspirillum seropedicae ZJUCS 1002 was 25.46%. Detailed implementation manners

[0021] The present invention will be further described below in conjunction with specific embodiments, but the protection scope of the present invention is not limited thereto:

[0022] Example 1: Isolation of the strain Herbaspirillum seropedicae ZJUCS 1002 of the genus Herbaspirillum

[0023] Nutrient agar medium (NA): The formula includes 10.0 g of peptone; 5.0 g of beef extract powder; 5.0 g of sodium chloride; 12.0 g of agar; pH = 7.3 g, and the volume is made up to 1000 mL with distilled water; the preparation method is to weigh 10.0 g of peptone and 5.0 g of beef extract powder, add 12 g of agar, continue to heat and stir evenly. After the agar is completely dissolved, add 5.0 g of sodium chloride, stir evenly, cool slightly and then make up the water to 1000 mL, and adjust the pH = 7.3. Dispense into test tubes or conical flasks, stopper and wrap. After sterilization at 1.1 atmospheres and 121 °C for 20 minutes, take it out and store it for later use after cooling.

[0024] The strain described in the present invention can be obtained by separating and culturing under the following conditions in the laboratory.

[0025] The strain Herbaspirillum seropedicae ZJUCS 1002 was isolated from the leaves of the Longjing 43 variety tea tree in the Tea Science Experimental Base of Zhejiang University in Hangzhou, Zhejiang Province (30.39°N, 119.88°E). The collected leaf samples were taken back to the laboratory and rinsed under tap water to remove surface impurities. After rinsing, surface disinfection was carried out.

[0026] After surface disinfection, an appropriate amount of plant material was placed in a sterile mortar and ground into a homogenate, and then serially diluted with sterile water. The diluted homogenate was spread on the NA medium and placed in an incubator for 24 hours. Single colonies were picked in a sterile laminar flow bench and streak-separated three times to obtain a pure culture.

[0027] Through a plate confrontation experiment, the endophytic bacterium ZJUCS 1002, which has a growth inhibitory effect on Colletotrichum camelliae, was selected as the Herbaspirillum seropedicae of the present invention. It is a Gram-negative bacterium. After incubation at 25 °C for 48 hours on NA medium, the bacterium forms an opaque milky white round raised colony with a diameter of 1-2 mm. The center of the colony bulges and the edge is neat.

[0028] The bacterium ZJUCS 1002 is a strain of the genus Herbaspirillum, and the preservation information is as follows: The preservation name is: Herbaspirillum seropedicae, the preservation unit: China General Microbiological Culture Collection Center, the preservation address: No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, the preservation date: April 19, 2022, the preservation number: CGMCC NO: 24723.

[0029] Example 2. Identification of the Herbaspirillum seropedicae strain ZJUCS 1002

[0030] 1. Molecular identification:

[0031] 1) PCR amplification of the bacterial 16s rDNA gene

[0032] A 50 μL reaction system was used for PCR amplification, and the system contained: 2 μM each of upstream and downstream primers, 200 μM dNTPs, Mg 2+ 1.5 mM, 5 μL of 10×PCR buffer, 2 μL of bacterial solution, 2 U of Taq enzyme, and ddH2O was added to make up to 50 μL. The upstream primer 16S sequence is 5′-AGAGTTTGATCMTGGCTCAG-3′, and the downstream primer sequence is 5′-TACGGYTACCTTGTTACGACTT-3′. The PCR amplification reaction was carried out on a BIORAD S1000 type PCR instrument.

[0033] Note: The above primers are the universal primers 27f and 1492r for bacterial 16s identification.

[0034] Reaction conditions: Pre-denaturation at 95 °C for 5 min, and then 35 cycles including: denaturation at 95 °C for 30 sec, annealing at 55 °C for 30 sec, extension at 72 °C for 1 min. Finally, post-extension at 72 °C for 7 min.

[0035] 2) Recovery and purification of the PCR product:

[0036] After the PCR reaction, the PCR products were detected by 1% agarose gel electrophoresis and then purified using the DNA Gel Extraction Kit from Axygen Biotechnology Company according to the steps in the kit's instruction manual.

[0037] The steps are as follows:

[0038] (1) After electrophoresis, cut out the gel containing the target DNA fragment with a blade under ultraviolet light and place it in a 2 mL centrifuge tube, then weigh it.

[0039] (2) Add 3 volumes of DE-A buffer and incubate at 75 °C for 10 min, during which shake several times until completely melted.

[0040] (3) Add 0.5 volumes of DE-B buffer and mix well.

[0041] (4) Place the DNA preparation tube in a 2 mL centrifuge tube, transfer the mixture to the DNA preparation tube, centrifuge at 12000 rpm for 1 min, and discard the supernatant.

[0042] (5) Place the DNA preparation tube back into the 2 mL centrifuge tube, add 500 μL of buffer W1, and centrifuge at 12000 rpm for 30 s.

[0043] (6) Place the DNA preparation tube back into the 2 mL centrifuge tube, add 700 μL of buffer W2, and centrifuge at 12000 rpm for 30 s.

[0044] (7) Repeat step (6) once.

[0045] (8) Place the DNA preparation tube back into the 2 mL centrifuge tube and centrifuge at 12000 rpm for 2 min to drain the washing solution on the membrane.

[0046] (9) Place the DNA preparation tube back into the 2 mL centrifuge tube, add 50 μL of ddH2O, centrifuge at 10000 rpm for 1 min, and elute the DNA and store it at -20 °C.

[0047] 3) Sequencing and sequence analysis of the gene

[0048] The purified and recovered target DNA fragment was sent to Sangon Biotech (Shanghai) Co., Ltd. for sequencing using an ABI PRISMA 377 automated sequencer after electrophoresis detection. After the sequencing results were strictly verified, a 1403 bp DNA fragment sequence (SEQ ID NO: 1 in the sequence listing) was obtained. The measured nucleotide sequence (SEQ ID NO: 1) was searched and aligned with homologous or similar nucleotide sequences in GenBank using BLAST. Based on the database annotations of the homologous sequences and combined with the morphological structure of the strain, the genus and species of the studied strain were determined.

[0049] After BLAST alignment, the sequence has a 100% coverage rate and 99.93% similarity with the sequence with the accession number HQ219859.1, and a 100% coverage rate and 99.86% similarity with the sequence with the accession number AY486380.1. By comparison, it is found that the sequence has a relatively high coverage rate with the two sequences.

[0050] This bacterium was identified as a strain of the genus Herbaspirillum and was preserved as follows: The preservation name is Herbaspirillum seropedicae ZJUCS 1002, the preservation address is Institute of Microbiology, Chinese Academy of Sciences, Beijing, China; the preservation date is April 19, 2022, and the preservation number is CGMCC NO: 24723. The strain ZJUCS 1002 in the present invention has the following taxonomic status: Bacteria, Proteobacteria, Betaproteobacteria, Burkholderiales, Oxalobacteraceae, Herbaspirillum.

[0051] Example 3: Inhibitory effect of Herbaspirillum seropedicae ZJUCS 1002 on the mycelial growth of Colletotrichum camelliae

[0052] Note: During the strain isolation process of Example 1, a control strain of Herbaspirillum huttiense was also obtained, and this strain has an inhibitory effect on the mycelial growth of Colletotrichum camelliae. Using the same method as in Example 2, based on 16S rDNA gene sequencing identification, it was determined to belong to Herbaspirillum huttiense.

[0053] 1) Preparation of Colletotrichum camelliae fungal cakes

[0054] The Colletotrichum camelliae that had been activated on a slant was inoculated onto a PDA plate medium and cultured in a constant temperature incubator at 25°C for 7 days. Then, a 7-mm sterile punch was used to punch holes to obtain fungal cakes.

[0055] 2) Preparation of the fermentation broth of Herbaspirillum seropedicae ZJUCS 1002 strain and Herbaspirillum huttiense control strain

[0056] The cryopreserved Herbaspirillum seropedicae ZJUCS 1002 strain and the Herbaspirillum huttiense control strain were taken out and spread on the NA medium plate for 24 h of culture (the culture temperature was 25 °C). Single colonies were picked and streaked on the NA medium plate for 24 h. Then, single colonies were picked and inoculated into the NA liquid medium, and cultured with shaking at 25 °C and 150 rpm for 24 h to obtain the strain fermentation broth.

[0057] 3) Plate confrontation test

[0058] The tea tree anthracnose fungal cake was placed in the center of the PDA plate medium, and sterile filter papers were placed 25 mm on both sides. 10 μL of the fermentation broth of the Herbaspirillum seropedicae ZJUCS 1002 strain and the Herbaspirillum huttiense control strain was added dropwise to the filter papers. The plate without added fermentation broth was used as a control. It was incubated at a constant temperature of 25 °C for 10 d, the mycelial diameter was measured, and the inhibition rate was calculated.

[0059]

[0060] As can be seen from Table 1, the Herbaspirillum seropedicae ZJUCS 1002 strain and the Herbaspirillum huttiense control strain had a significant inhibitory effect on the growth of anthracnose fungal hyphae. Among them, the inhibition rate of the Herbaspirillum seropedicae ZJUCS 1002 strain reached 34.44%, while the inhibition rate of the Herbaspirillum huttiense control strain was 25.82%.

[0061] Table 1 Inhibitory effect of ZJUCS 1002 strain on anthracnose fungus

[0062]

[0063]

[0064] The above experiments were repeated three times to obtain the plate confrontation of the Herbaspirillum seropedicae ZJUCS 1002 strain and the Herbaspirillum huttiense control strain with anthracnose fungus ( Figure 1) Experimental results showed that the strain Herbaspirillum seropedicae ZJUCS 1002 had a significant inhibitory effect on the growth of Colletotrichum camelliae hyphae, and was significantly superior to the control strain Herbaspirillum huttiense.

[0065] Example 4: Effect of Herbaspirillum seropedicae ZJUCS 1002 strain on controlling Colletotrichum camelliae on tea leaves

[0066] 1) Preparation of conidial suspension of Colletotrichum camelliae on tea plants

[0067] Inoculate the Colletotrichum camelliae on the PDA plate medium after slant activation, and culture it in a constant temperature incubator at 25 °C for 30 days. Add 10 ml of sterile water to the PDA plate, scrape the mycelium and conidia with a sterile spreader, and filter through three layers of sterile lens paper to obtain the conidial suspension. After examining the concentration under the microscope, add 0.1% (v / v) Tween-80 and adjust its concentration to 1×10 5 conidia / ml of the conidial suspension.

[0068] 2) Preparation of fermentation broth of Herbaspirillum seropedicae ZJUCS 1002 strain and Herbaspirillum huttiense control strain

[0069] Take out the Herbaspirillum seropedicae ZJUCS 1002 strain and the Herbaspirillum huttiense control strain stored at low temperature, coat and culture them on the NA medium plate for 24 h. Pick single colonies and streak them on the NA medium plate for 24 h. Then pick single colonies and inoculate them into the NA liquid medium, and shake culture at 25 °C and 150 rpm for 24 h to obtain the strain fermentation broth, and adjust the concentration to 1×10 8 cfu / ml for inoculation.

[0070] 3) Effect of Herbaspirillum seropedicae ZJUCS 1002 strain and Herbaspirillum huttiense control strain on reducing the control effect of Colletotrichum camelliae on tea leaves

[0071] Pick the fresh young leaves of healthy two-year-old cuttings of the Longjing 43 tea tree variety with similar size and tenderness. After soaking in 1% sodium hypochlorite solution for 4 minutes, wash them with sterile water for surface disinfection. After the leaves are air-dried until there is no obvious water stain on the surface, use an atomizer to spray the fermentation broth of Herbaspirillum seropedicae ZJUCS 1002 strain and Herbaspirillum huttiense control strain at 1×10 8 cfu / mL, with sterile water as the control, and spray until dripping; place the leaves in a square petri dish. After placing them in the dark at 25°C for 6 hours, use a sterile needle to pierce 12 holes on both sides of the middle vein of the leaves, and use an atomizer to spray the conidia solution of Colletotrichum fungi on the leaves. Place the leaves in a climate chamber at 25°C. After 24 hours in the dark, set the photoperiod of the climate chamber to 14h / 10h, and the light intensity to 10000 lx. After co-culturing for 7 days, isolate and identify the lesion positions on the tea trees, and obtain the tea tree anthracnose strains consistent with the inoculated Colletotrichum camelliae. Take out the leaves and take pictures, use ImageJ software to calculate the lesion area of the leaves, and calculate the incidence of anthracnose on tea tree leaves.

[0072]

[0073] As can be seen from Table 2, both the Herbaspirillum seropedicae ZJUCS 1002 strain and the Herbaspirillum huttiense control strain have significant control effects on anthracnose of tea tree leaves. Among them, the incidence of anthracnose in the control group is 56.47%, while the incidence of anthracnose on tea tree leaves after inoculation with Herbaspirillum seropedicae ZJUCS 1002 is only 25.46%, effectively reducing the incidence of anthracnose on tea tree leaves by 31.01%. The incidence of anthracnose on tea tree leaves inoculated with the Herbaspirillum huttiense control strain is 39.16%, only reducing the incidence of anthracnose on tea tree leaves by 17.31%.

[0074] Table 2 Control effect of ZJUCS 1002 strain on anthracnose of tea tree leaves

[0075]

[0076]

[0077] Figure 3The results showed that both the Herbaspirillum seropedicae ZJUCS 1002 strain and the Herbaspirillum huttiense control strain had good control effects on anthracnose of tea leaves, with the control effect of the Herbaspirillum seropedicae ZJUCS 1002 strain being 54.9% and that of the Herbaspirillum huttiense strain being 30.6%. Therefore, Herbaspirillum fungi had obvious effects on anthracnose of tea trees, and the control effect of the Herbaspirillum seropedicae ZJUCS 1002 strain was significantly better than that of the Herbaspirillum huttiense control strain.

[0078] Finally, it should be noted that the above examples are only some specific embodiments of the present invention. Obviously, the present invention is not limited to the above embodiments, and there are many variations. All variations that can be directly derived or associated with the content disclosed by a person skilled in the art should be considered as the protection scope of the present invention.

Claims

1. Herbaspirillum seropedicae ( Herbaspirillum seropedicae ), strain ZJUCS 1002, characterized in that the deposit number is: CGMCC NO: 24723.

2. Use of the Herbaspirillum seropedicae strain ZJUCS 1002 as described in claim 1, characterized in that: Prevention and control of Colletotrichum camelliae Massee Colletotrichum camelliae which causes anthracnose of tea plants.

3. Use of the Herbaspirillum seropedicae strain according to claim 2, characterized in that: Inhibiting Colletotrichum camelliae Colletotrichum camelliae Hyphal growth.

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