Biocontrol strain G101 for preventing and treating litchi downy blight and application of biocontrol strain G101

Abstract

The invention discloses a biocontrol strain G101 for preventing and treating litchi downy blight and application of the biocontrol strain G101. The Acinetobacter Pittii G101 for preventing and treating the litchi downy blight is preserved in the Guangdong Microbiological Culture Collection Center on May 28, 2024, and the preservation number of the Acinetobacter Pittii G101 is GDMCC No.64696. The Acinetobacter Pittii G101 can be used for preventing and treating the litchi downy blight. The biocontrol strain G101 is applied to prevention and treatment of litchi peronophythora blight and preservation of litchis. The acinetobacter picolini obtained by the invention is derived from litchi pericarp bacteria; and because of long-term coexistence and mutualistic symbiosis with litchis, litchi pericarp microorganisms are not influenced, and when being sprayed to the surfaces of the litchis for preventing and treating diseases, the litchis are safer and more reliable to eat and processed products of the litchis.

Description

Technical Field

[0001] The invention belongs to the technical field of plant protection and relates to a biocontrol strain G101 for preventing and controlling litchi downy blight and an application thereof. Background Art

[0002] Litchi (Litchi chinensis Sonn) is an important subtropical cash crop in southern China. It is known as the "precious fruit of China". It has high economic value and is one of the most competitive fruits in my country in the international market. After being harvested, litchi fruits tend to turn brown and rot in normal or low-temperature cold storage. The main diseases that cause fruit rot and deterioration include litchi anthracnose, sour rot, and downy mildew. Among them, downy mildew caused by Peronophythora litchii Chen ex Ko et al. is the most serious (Kao et al., 1980; Qi Peikun et al., 1984). The fungus likes high humidity and can infect young leaves, flower spikes, young fruits, branches, and fruit stalks, causing flowers and fruits to fall off; it prefers to infect nearly mature fruits. During transportation, if any fruit carries pathogens, they will all become diseased, rot and deteriorate, causing great economic losses (Xu et al., 2013; Cai Yongchun, 2008; Liang Gaijin et al., 2001; Yang Dedong, 2015). When the humidity is high, white mold appears on the surface of the fruit, namely sporangia and sporangiophores (Cai Xueqing, 2010). At present, it occurs in several major hotspots, but the incidence rate in Yunnan Province is relatively low (Zhang Rong, 2012). At present, the effective measures to control litchi frost disease are mainly chemical control, but the large-scale use of chemical pesticides may induce environmental pollution and pathogen resistance problems are becoming increasingly prominent. Therefore, research and development of low-toxic, residue-free bio-source pesticides for litchi disease control is of great significance for controlling pesticide residues, ensuring food safety and improving product international competitiveness.

[0003] Acinetobacter sp. is a common bacterium that is widely present in nature. It has many important ecological functions and antagonistic effects on some diseases. 20210508.X discloses an Acinetobacter Pittii that has a bioremediation effect on soil. It can effectively regulate soil microorganisms and can be used for the treatment of petroleum wastewater, petroleum-contaminated soil, etc. It not only has a high degradation rate but also is easy to operate. 202310722523.6 discloses a strain of Acinetobacter LH-12 that can significantly promote corn growth, increase soil nutrients, and convert phosphorus. 201811084608.1 discloses a strain of Acinetobacter JQ1004, which can nitrify with organic carbon as a carbon source under the same aerobic conditions to provide nutrients as a nitrate and nitrogen source to promote plant growth. At present, there are few relevant literatures on biological control in the application of Acinetobacter. Summary of the invention

[0004] The purpose of the present invention is to provide a biocontrol strain G101 for controlling litchi downy blight in view of the above-mentioned deficiencies in the prior art.

[0005] Another object of the present invention is to provide application of the strain.

[0006] Another object of the present invention is to provide a bacterial agent containing the strain and a preparation method thereof.

[0007] The purpose of the present invention can be achieved through the following technical solutions:

[0008] An Acinetobacter Pittii G101 for preventing and controlling litchi frost blight was deposited in the Guangdong Provincial Microbiological Culture Collection Center on May 28, 2024, and the culture collection number is GDMCC No.64696.

[0009] A biocontrol agent prepared from the Acinetobacter Pittii G101.

[0010] The application of Acinetobacter Pittii G101 in preparing products for preventing and treating litchi downy blight.

[0011] The application of Acinetobacter Pittii G101 in preventing and controlling litchi downy blight.

[0012] As a preferred embodiment of the present invention, the Acinetobacter piezoelectrica G101 with the accession number of GDMCC No.64696 is used as the active ingredient.

[0013] As a preferred embodiment of the present invention, the biocontrol agent is prepared by the following method: prepared by liquid fermentation of the Acinetobacter piezoelectrica G101, specifically comprising the following steps: streaking the Acinetobacter piezoelectrica G101 on an LB solid culture medium, placing it in a 28°C incubator for dark culture for 24 hours; picking a single colony to prepare a seed solution in an LB culture solution, placing it at 28°C and 200r / min for shaking culture for 8-12 hours; taking a certain amount of the prepared seed solution (the amount taken is 1%-5% of the LB culture solution) and transferring it to the LB culture solution to prepare a fermentation solution, placing it at 28°C and 200r / min for shaking culture for 18-20 hours.

[0014] The biocontrol fungus agent is used in preparing products for preventing and controlling litchi downy blight.

[0015] The application of the biocontrol agent in preventing and controlling litchi downy blight.

[0016] Beneficial effects of the present invention:

[0017] The Acinetobacter piezogenes obtained by the present invention is isolated from litchi peel. Since it is isolated and obtained on the surface of litchi fruit, it is foreseeable that it can coexist with litchi for a long time, mutually benefit and coexist, and form a series of survival mechanisms similar to litchi. Spraying it on the surface of litchi for disease prevention and control is safe and reliable for litchi consumption and litchi processed products.

[0018] The Acinetobacter piezogenes of the present invention has an antibacterial effect on litchi downy mildew, has an excellent biocontrol effect on litchi downy mildew, and is a biocontrol agent specially developed for litchi downy mildew. Since it is a biological agent, it does not have a series of problems caused by the use of chemical pesticides, and is therefore beneficial to the pollution-free production of litchi, and farmers do not need to use or reduce the use of other chemical pesticides, which not only saves farmers money but is also beneficial to the export of fruits.

[0019] The culture conditions of the Acinetobacter pievomitoris obtained by the present invention are easy to obtain, have great commercial potential, and have good development and application prospects.

[0020] Biological sample deposit information

[0021] Acinetobacter pittii G101, classified as Acinetobacter Pittii, was deposited in the Guangdong Provincial Microbiological Culture Collection Center on May 28, 2024. The collection address is 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou, Guangdong Institute of Microbiology, and the culture collection number is GDMCC No: 64696. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Figure 1 Single colony image of biocontrol bacteria G101

[0023] Figure 2 Effects of three different components (supernatant, bacterial suspension, and fermentation broth) of biocontrol bacteria G101 on mycelial growth

[0024] Figure 3 Effects of three components of strain G101 on sporangium germination of litchi downy mildew; Sporangium germination status of litchi downy mildew

[0025] Figure 4 Effects of three components of strain G101 on zoospore germination of Lychee Downy Phytophthora; Germination status of zoospore of Lychee Downy Phytophthora

[0026] Figure 5 Lychee fruit and leaf disease rating

[0027] Figure 6Results of the treatment group and the biocontrol group Analysis of the biocontrol effect of G101 on litchi downy mildew Note: a. Disease index and biocontrol effect of 'Guiwei' on litchi leaves; b. Disease index and biocontrol effect of 'Guiwei' on litchi fruits;

[0028] c. Disease index and preventive effect of litchi fruit 'Fei Zi Xiao'.

[0029] Figure 7 Litchi fruit and leaf prevention effect diagram DETAILED DESCRIPTION

[0030] Example 1

[0031] Source of strain G101: G101 was isolated from the peel of 'Guiwei' lychee in a lychee orchard in Guangzhou, Guangdong Province.

[0032] Samples were collected from a litchi forest in Guangdong Province in July 2020. Litchi fruits pretreated with AF (α-Farnesene, AF, 100 mg / L) were inoculated with P. litchii (5×10 4 The samples collected after the onset of disease in the control group were quickly put into numbered plastic bags and taken back to the laboratory for separation. The method was to cut 3g of peel and add it to 27mL of sterile water (containing sterile glass beads), shake it on a shaker at 180rmp for 0.5h, let it stand for 5min, and get 10 -1 Dilution, take 0.1mL of supernatant and dilute it successively, take 10 -4 , 10 -5 , 10 -6 Three gradients of 0.1 mL were coated with R2A (R2A medium preparation method: yeast powder 0.5 g, casein peptone 0.25 g, meat peptone 0.25 g, hydrolyzed casein 0.5 g, glucose 0.5 g, starch 0.5 g, pyruvic acid 0.3 g, potassium dihydrogen phosphate 0.3 g, hydrated magnesium sulfate 0.024 g, agar 15 g, made up to 1 L with ultrapure water, adjusted pH ≈ 7.2, sterilized at 121°C for 20 min), cultured at 28°C for 48-72 h, selected plates with colony counts between 50 and 300, counted and picked colonies, purified, stored at -80°C, 40% glycerol for later use. Among them, a strain of bacteria was found and named G101.

[0033] G101 colonies on culture plates are milky white, round, and have neat edges. The growth temperature range is 28-37°C. Bacterial gene DNA was extracted using a bacterial genome kit (Shanghai SBS Genetech Co. Ltd) and then amplified by BOX-PCR. The 16S rRNA gene fragment was amplified from genomic DNA using the 16S rRNA universal primers U8-27 (F) 5'-AGA GTT TGA TC (AC) TGG CTC AG-3'; L1494-1514 (R) 5'-CTACGG (AG) TACCT TGT TAC GAC-3', purified and connected to the pMD19-T vector, transformed into Escherichia coli DH5α, extracted positive clone plasmids, commissioned TaKaRa Company for sequencing, and compared at NCBI (National Center for Biotechnology Information) to obtain identification information. The bacterium was identified as Acinetobacter Pittii by sequencing of the 16S rDNA gene fragment sequence (Table 1).

[0034] Table 1 Sequencing alignment results

[0035]

[0036] Example 2 Preparation of bacterial agent

[0037] (1) Cultivation of seed culture liquid: G101 (GDMCC No. 64696) was inoculated into LB culture liquid (LB culture medium preparation method: sodium chloride 10g, tryptone 10g, yeast extract 5g, agar 15g, liquid culture liquid without agar, made up to 1L with ultrapure water, adjusted to pH ≈ 7.2, sterilized at 121°C for 20min), cultured at 28°C, 200rpm shaking for 24h, and sampled every 3h in an ultra-clean bench after 16h to measure OD 600 During the measurement, the uninoculated culture medium was used to adjust the zero value until the seed culture OD 600 When the pH is 0.6, the culture is terminated and the seed bacterial solution is obtained;

[0038] (2) Fermentation broth preparation: G101 (GDMCC No. 64696) seed solution was inoculated into 20 mL LB culture medium at a volume ratio of 1:10, and cultured at 28°C and 200 rpm for 12 h. The OD value of the culture solution was adjusted. 600 is 0.98-1.07, and a fermentation liquid is obtained;

[0039] (3) Preparation of bacterial suspension: After obtaining the fermentation broth, centrifuge it at 4°C, 8000 rpm for 15 min, discard the supernatant, and resuspend the bacteria with sterile water. Adjust the OD 600About 1, to obtain a bacterial suspension;

[0040] (4) Preparation of supernatant: After obtaining the fermentation broth, centrifuge the fermentation broth at 4°C and 8000 rpm, and filter the supernatant with a 0.22 μm filter membrane to obtain a sterile supernatant;

[0041] (5) Preparation of sporangium suspension: 10 mL of sterile water was added to the pathogen culture dish covered with mycelium, and the dish was thoroughly scrubbed with a sterile brush. The mycelium was filtered through a sterile tea strainer into a sterilized conical flask, and 1 μL of the filtrate was dropped onto a glass slide. The sporangium was counted under a microscope to obtain a final concentration of 50 / μL, thus obtaining a sporangium suspension (5×10 4 sporangium / mL).

[0042] (6) Preparation of zoospore suspension: The sporangium suspension was pretreated with low temperature to stimulate the release of zoospores. It was first placed in a 4°C refrigerator for 1 h, then placed at 25°C for 1 h, and finally placed in a 4°C refrigerator for 1 h. After being taken out, it was allowed to stand for 10 min to allow the sporangium to settle at the bottom of the bottle. The supernatant was aspirated and filtered through a 0.20 μm filter membrane to obtain a pure zoospore suspension. 1 μL of the filtrate was dropped on a glass slide and the zoospores were counted under a microscope to obtain a final concentration of 50 zoospores / μL, i.e., a zoospore suspension (5×10 4 zoospores / mL).

[0043] Example 3 Effects of the three components of biocontrol bacteria G101 on the mycelial growth of Downy mildew

[0044] The confrontation culture method was used to inoculate the downy mildew fungus stored at 20°C into CJA medium (CJA medium preparation method: 200g fresh carrots were peeled and chopped, water was added and juiced for 10 minutes, the juice was filtered after juicing, 15g agar was added to the filtrate, the volume was made up to 1L with ultrapure water, the pH was adjusted to 7.2, and sterilized at 121°C for 20 minutes) for activation. After the fungus grew all over the plate, a sterilized punch was used to evenly punch the outer edge of the colony into round fungus blocks with a diameter of 5mm. The mycelium block of Downy Phytophthora was inoculated in the center of a 1:1 volume mixed CA-PDA medium (PDA medium preparation method: 200g fresh potatoes were peeled and chopped, boiled with water, and pierced with chopsticks. After filtering, 20g glucose and 15g agar were added, and the volume was fixed to 1L with ultrapure water, and the pH was adjusted to 7.2, and sterilized at 121°C for 20min). The G101 supernatant, bacterial suspension and fermentation liquid prepared in Example 2 were inoculated at about 27mm from the center around it, and four replicates were used for each treatment. The plate was cultured at 28°C for 7 days, and the mycelium growth diameter was recorded after the control mycelium covered the plate.

[0045] The experimental data showed that the three components of G101 had different effects on mycelial growth. The supernatant had no inhibitory effect, while the inhibitory effects of the fermentation broth and bacterial suspension were 38.5% and 58.9%, respectively (Table 2).

[0046] Table 2 Inhibitory effect of biocontrol bacteria G101 on mycelial growth of litchi peronophyte

[0047]

[0048] Example 4 Effects of the three components of biocontrol bacteria G101 on the germination of sporangia of Downy mildew

[0049] Take 5mL of the prepared culture medium (i.e., add 0.5mL of G101 supernatant / bacterial suspension / bacterial suspension to 4.5mL of sporangium suspension, and add 0.5mL of sterile water to the control group) and put it into a 15mL centrifuge tube. Place the centrifuge tube at 28℃ and 180rpm for shaking culture. Culture for 6h and 8h, and take the mixed culture medium for observation. Each treatment group was repeated 3 times, and 3 fields of view were taken for each repeat. 50-60 sporangia were observed in each field of view (germination was considered as germ tube length greater than or equal to sporangium length and empty sporangium) (Xing Mengyu, 2017).

[0050] Sporangium germination rate = number of sporangium germination / total number of sporangia × 100%

[0051] Sporangium germination inhibition rate = (germination rate of control group - germination rate of treatment group) / germination rate of control group × 100%

[0052] The results showed that the three components of strain G101 (supernatant, bacterial suspension and fermentation broth) all had an inhibitory effect on the germination of conidia of Peronophytum litchii, with inhibition rates of 45.6%, 29.7% and 61.2%, respectively (Table 3, Figure 3 )

[0053] Table 3 Inhibitory effect of biocontrol bacteria G101 on spore germination of Lychee Downy Phytophthora

[0054]

[0055] Example 5 Effects of the three components of biocontrol bacteria G101 on zoospore germination of Downy mildew

[0056] Take 5mL of the prepared culture medium (i.e., add 0.5mL of G101 supernatant / bacterial suspension / bacterial suspension to 4.5mL of zoospore suspension, and add 0.5mL of sterile water to the control group) and put it into a 15mL centrifuge tube. Place the centrifuge tube at 28℃ and 180rpm for shaking culture. Culture for 3h and 5h, and take the mixed culture medium for observation. Each treatment group was repeated 3 times, and 3 fields of view were taken for each repeat. 50-60 zoospores were observed in each field of view (germination was considered to be when the length of the germ tube was greater than or equal to the diameter of the zoospore) (Xing Mengyu, 2017).

[0057] Zoospore germination rate = number of zoospores germinated / total number of zoospores × 100%

[0058] Zoospore germination inhibition rate = (germination rate of control group - germination rate of treatment group) / germination rate of control group × 100%

[0059] The results showed that the three components of strain G101 (supernatant, bacterial suspension and fermentation broth) all had an inhibitory effect on the germination of zoospores of Peronophytum litchii, with inhibition rates of 50.7%, 27.83% and 65.42% respectively. (Table 4, Figure 4 )

[0060] Table 4 Inhibitory effect of biocontrol bacteria G101 on zoospore germination of litchi peronophyte

[0061]

[0062]

[0063] Example 6 Evaluation of the in vitro efficacy of biocontrol bacteria G101 on litchi leaves and fruits

[0064] Healthy litchi fruits / leaves picked from the field (the litchi varieties in this embodiment are 'Guiwei' and 'Feizixiao') were treated as follows: A. Biocontrol bacteria suspension stock solution (bacterial suspension OD 600 1) dilute 50 times to spray on leaves, dilute 10 times to spray on fruits; B. Sterile water with the same dilution multiple. Each treatment has 3 replicates, each replicate has 5 branches / 30 fruits, and the branches / fruits are placed in a fresh-keeping box (the bottom of the box is covered with sterile filter paper, with a diameter of 18 cm, moistened with sterile water, the specifications of the fresh-keeping box are 323*220*100mm, and the manufacturer is Hualong Plastic Factory, Chancheng District, Foshan City). Spray 300mL of the bacterial solution and the control group for each treatment (3 replicates). Spray the inoculated pathogen P. litchii (5*10 4 The disease index was investigated at 36 hpi (hours post inoculation) and the biocontrol effect was calculated.

[0065] Disease investigation method (Cai Xueqing, 2010): Disease level diagram as follows Figure 5 As shown. Grade 0: no symptoms; Grade 1: slight lesions at the inoculation point, necrotic area less than 5%; Grade 3: necrotic area 5%-10%; Grade 5: necrotic area 10%-25%; Grade 7: necrotic area 25%-50%; Grade 9: necrotic area exceeds 50% or all white mold (fruit) or browning (leaves). Statistical calculation method of disease index and biocontrol effect: disease index (%) = [∑ (number of disease levels × number of fruits of the disease level) / (highest disease level × total number of fruits)] × 100; biocontrol effect (%) = [(control disease index - treatment group disease index) × 100 / control disease index].

[0066] The biocontrol effect analysis of this experiment includes: 1. Pick healthy litchi leaves ('Guiwei'), spray them with G101 suspension in the laboratory after pruning, spray and inoculate litchi downy mildew 24 hours later, and then observe the disease index. 2. Pick healthy litchi fruits ('Guiwei' and 'Feizixiao') with a maturity of about 85%, spray them with G101 suspension in the laboratory, spray and inoculate litchi downy mildew 24 hours later, and then observe the disease index.

[0067] The results showed that in the detached leaf 'Guiwei' experiment, at 84 and 108 hpi, the protective effects of G101 on litchi leaf 'Guiwei' were 37.8% and 42.2%, respectively, with an average protective effect of 33.75% (Table 5, Figure 6 a, Figure 7 ).

[0068] Table 5 Analysis of the protective effect of strain G101 on LDB in litchi leaves (2022)

[0069]

[0070] x Biocontrol bacteria dilution (1×10 7 CFU / mL) spray pretreatment of litchi leaves in indoor fresh-keeping boxes'Guiwei'; 24h spray inoculation of pathogenic bacteria P. litchii (5×10 4 Sporangium / mL). Z AE, indicates the average control effect. The data are the average of three replicates, and the letters after the data indicate significant differences between treatments at the 95% level (Duncan's test).

[0071] In the in vitro fruit experiment, G101 showed good control effect on litchi fruit. On 'Guiwei', the disease index of the control group at 108 hpi was 75.8±8.68%, and that of the G101-treated group was 52.3±3.42%. The control effect after G101 treatment was 35.2%, and the average control effect at the two time points was 33.75% (Table 6, Figure 6 b, Figure 7); On 'Fei Zi Xiao', the disease index of the control group at 84 hpi was 34.94±8.50%, and that of the G101-treated group was 16.30±0.98%. The protective efficacy after G101 treatment was 53.4%, and the average protective efficacy at the two time points was 39.85% (Table 7, Figure 6 c, Figure 7 ).

[0072] Table 6 Analysis of the biocontrol effect of biocontrol bacteria G101 on downy mildew of 'Guiwei' fruit in vitro (2023)

[0073]

[0074] hpi (hours post inoculation), hours after inoculation of pathogens.

[0075] x1 Litchi fruits of 'Guiwei' (85% maturity, picked from the field and stored in a laboratory fresh-keeping box) were spray-pretreated and incubated at room temperature (25°C). The pathogen P. litchii (5×10 4 sporangium / mL). x2 Biocontrol bacteria G101 suspension (5×10 7 Litchi fruit 'Guiwei' was pretreated by spraying with 5×10 4 sporangium / mL). x3 Litchi fruits were pretreated with sterile water spray, cultured at room temperature (25°C), and spray-inoculated with pathogenic bacteria P. litchii (5×10 4 sporangium / mL). Z AE, average efficacy, means average efficacy. The data are the average of three replicates, and the letters after the data indicate significant differences between treatments at the 95% level (Duncan's test).

[0076] Table 7 Analysis of the biocontrol effect of biocontrol bacteria G101 on detached fruit of 'Fei Zi Xiao' (2024)

[0077]

[0078] The method is the same as Table 6.

Claims

1. An Acinetobacter Pittii G101 for preventing and controlling litchi frost blight was deposited in the Guangdong Provincial Microbiological Culture Collection Center on May 28, 2024, and the culture collection number is GDMCC No.64696.

2. Use of Acinetobacter Pittii G101 according to claim 1 in preparing products for preventing and treating litchi downy blight.

3. Use of AcinetobacterPittiiG101 according to claim 1 in preventing and treating litchi downy blight.

4. A biocontrol agent prepared from Acinetobacter Pittii G101 according to claim 1.

5. The biocontrol agent according to claim 4, characterized in that The active ingredient is Acinetobacter Pittii G101 with the deposit number of GDMCC No. 64696 as claimed in claim 1, wherein the viable bacterial concentration of Acinetobacter Pittii G101 is 1×10 5 ~10 6 CFU / mL.

6. The biocontrol agent according to claim 4, characterized in that The biocontrol agent is prepared by the following method: the biocontrol agent is prepared by liquid fermentation of Acinetobacter Pittii G101, which specifically comprises the following steps: inoculating Acinetobacter Pittii G101 into LB culture solution and culturing at 28-30°C and 180-200rpm for 20-30h to obtain the biocontrol agent.

7. Use of the biocontrol agent according to any one of claims 4 to 6 in the preparation of a product for preventing and controlling litchi downy blight.

8. Use of the biocontrol agent according to any one of claims 4 to 6 in controlling litchi downy blight.

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