Leuconostoc mesenteroides and application thereof in prevention and treatment of postharvest apple fruit diseases

Abstract

The invention relates to the field of biological prevention and control of postharvest diseases of fruits, in particular to leuconostoc mesenteroides GY-2 and application thereof in prevention and control of postharvest apple fruit diseases. The Leuconostoc mesenteroides GY-2 is preserved, and the preservation number of the Leuconostoc mesenteroides GY-2 is CGMCC (China General Microbiological Culture Collection Center) No.29733; and diluting the thalli into 108 CFU / mL bacterial suspension, inoculating the bacterial suspension to wounds on the surfaces of the fruits, inoculating pathogenic bacteria after the bacterial suspension is air-dried, and culturing at 25 DEG C. Compared with control treatment, the leuconostoc mesenteroides has different degrees of control effects on the three apple fruit diseases. Meanwhile, the strain can be colonized on the surfaces of apple fruits for a long time. The strain provides a new postharvest disease control and preservation scheme for controlling postharvest apple diseases.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of biological control of postharvest diseases of apples, and in particular to Leuconostoc mesenteroides and its use in the control of postharvest diseases of apples. BACKGROUND

[0002] Apple is a delicious fruit that contains various nutrients beneficial to humans. Apple plays an important role in the economy of China, and the apple planting area and yield in China ranks first in the world, being one of the largest apple producing areas in the world. The annual yield of apples in China is more than 500 million tons. However, the supply of apples is mainly affected by various fungal diseases during transportation, storage and sale, which affects the healthy development of the apple industry.

[0003] Postharvest diseases of apples include Alternaria alternata caused black spot disease, Botryospaeria dothidea caused red rot disease, and Colletotrichum gloeosporioides caused anthracnose. The pathogenic bacteria of the above three diseases can all infect from the natural orifices such as the surface wounds, microcracks, and skin pores of apples under the conditions of suitable environmental temperature and humidity, causing the decay and deterioration of fruits, the inedible fruit pulp, and seriously affecting the quality of apples. Therefore, it is necessary to take safe and effective measures to control postharvest diseases of apples.

[0004] At present, the main method to control postharvest diseases of apples is to store the picked apples in a cold storage, which has the advantages of safety, non-toxicity and harmlessness, but requires high equipment. In addition, chemical fungicides can be sprayed to control apple diseases, but due to environmental pollution, influence on human health, and the generation of resistant strains, biological control can control diseases by beneficial microorganisms, which has the advantages of safety and environmental protection compared with chemical control methods.

[0005] Leuconostoc mesenteroides is an important species of Leuconostoc in lactic acid bacteria, gram-positive, widely exists on the surface of plants, can ferment sugars to produce a variety of acids and alcohol, has the ability of high acid production, oxidation resistance and antagonism of pathogenic bacteria, and is often used in fermented dairy products, silage, pickles and fruit wine. So far, there are still few reports on Leuconostoc mesenteroides for the prevention and control of plant diseases, and the biological characteristics, antibacterial spectrum and other characteristics of strains from different sources have obvious differences, with significant strain specificity. The applied invention patent CN113308408A discloses a Leuconostoc mesenteroides capable of producing bacteriocin, which is isolated from cane juice raw materials, and has very obvious antibacterial effect on several foodborne pathogenic bacteria such as Staphylococcus aureus, Escherichia coli and Salmonella; the applied invention patent CN114262666A discloses a Leuconostoc mesenteroides with good antagonistic activity against aquatic pathogenic bacteria; the strain of the applied invention patent CN116240136A is isolated from pickle juice, and the Leuconostoc mesenteroides strain has high antagonistic activity against Valsa mali and Valsa mali asiatica.

[0006] So far, there is no report on the application of Leuconostoc mesenteroides as a biocontrol agent for postharvest diseases of apples, so this patent intends to develop a biocontrol agent for the green control of postharvest diseases of apples. SUMMARY

[0007] The present application provides a Leuconostoc mesenteroides and its application in the prevention and control of postharvest diseases of apples.

[0008] The Leuconostoc mesenteroides GY-2 strain, with the Latin name Leuconostoc mesenteroides, was preserved in the China General Microbiological Culture Collection Center (CGMCC) on January 23, 2024, with the preservation number CGMCC No. 29733.

[0009] The culture or metabolite of the Leuconostoc mesenteroides GY-2 also belongs to the protection scope of the present application.

[0010] The culture can be a bacterial liquid obtained by Leuconostoc mesenteroides GY-2 in a liquid medium, containing bacterial bodies and their metabolites.

[0011] The bacterial liquid can significantly control the occurrence of important apple diseases such as apple black spot, apple red rot and apple anthracnose.

[0012] The liquid bacterial agent can be a bacterial liquid obtained by Leuconostoc mesenteroides GY-2 in a liquid medium MRS, or a Leuconostoc mesenteroides GY-2 suspension prepared with sterile water.

[0013] The application has the beneficial effects that the application provides Leuconostoc mesenteroides GY-2 with a preservation number of CGMCC No. 29733 and application thereof in preparation of a preparation for preventing and treating postharvest diseases of apple fruits, which not only enriches a Leuconostoc mesenteroides strain resource library and widens the application range of the field of preventing and treating plant diseases by Leuconostoc mesenteroides, but also provides a new solution for preventing and treating the postharvest diseases of apple fruits, i.e. Alternaria disease, red rot disease and anthracnose disease. BRIEF DESCRIPTION OF DRAWINGS

[0014] The following drawings are added to supplement the description of the embodiments in the specific implementation.

[0015] Figure 1 Prevention and treatment effect of Leuconostoc mesenteroides GY-2 strain on apple black spot disease. The left graph is inoculated with sterile water and Alternaria alternata, and the right graph is inoculated with strain GY-2 bacterial suspension and Alternaria alternata.

[0016] Figure 2 Prevention and treatment effect of Leuconostoc mesenteroides GY-2 strain on apple red rot disease. The left graph is inoculated with sterile water and Stemphylium lycopersicon, and the right graph is inoculated with strain GY-2 bacterial suspension and Stemphylium lycopersicon.

[0017] Figure 3 Prevention and treatment effect of Leuconostoc mesenteroides GY-2 strain on apple anthracnose disease. The left graph is inoculated with sterile water and Colletotrichum gloeosporioides, and the right graph is inoculated with strain GY-2 and Colletotrichum gloeosporioides bacterial suspension.

[0018] Figure 4 Colonization dynamic diagram of Leuconostoc mesenteroides GY-2 on apple fruits.

[0019] Figure 5 Colony diagram of Leuconostoc mesenteroides GY-2.

[0020] Figure 6 Scanning electron microscope diagram of Leuconostoc mesenteroides GY-2.

[0021] Figure 7 Gram staining diagram of Leuconostoc mesenteroides GY-2.

[0022] Figure 8 Molecular identification diagram of Leuconostoc mesenteroides GY-2.

[0023] Biological preservation information

[0024] Leuconostoc mesenteroides, which is isolated from apple collected in the orchard of Hebei Agricultural University, is preserved in China General Microbiological Culture Collection Center, and the preservation time is January 23, 2024. The address is No. 3, Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences, the strain number is CGMCC No. 29733, and the strain number is GY-2. DETAILED DESCRIPTION

[0025] In order to make the purpose, technical scheme and advantages of the present application clearer, the embodiments of the present application will be further described below in combination with the drawings. The following examples facilitate better understanding of the present application, but do not limit the present application.

[0026] Example 1

[0027] Isolation, purification and screening of apple biocontrol bacteria.

[0028] A number of healthy Fuji apples were collected from the orchard of Hebei Agricultural University, surface sterilized with 1% sodium hypochlorite for 10 min, washed with sterile water three times, and 1 g of pulp and peel tissue of the apple fruit was collected with a sterile scalpel and ground thoroughly with 5 mL of sterile distilled water. 30 μl of the ground liquid was spread on PDA and incubated at 25°C for 3-5 days. Single colonies were isolated and suspended in 20% glycerol, and stored in a -80°C ultra-low temperature freezer.

[0029] Example 2

[0030] Disease prevention effect of Leuconostoc mesenteroides GY-2 strain on apple black spot, apple red rot and apple anthracnose.

[0031] A number of Fuji fruits were purchased from a local supermarket, and fruits with uniform size, uniform color, healthy surface and no wounds were selected. The surface was sterilized with 2% sodium hypochlorite solution for 1 min, and a needle was used to make a hole (4 mm x 4 mm) on the surface of the apple. 15 μl of bacterial suspension (10 8 CFU / mL) of strain GY-2 was injected into the hole with a pipette gun, and 15 μl of sterile water was injected as a blank control. 2 h after inoculation, 15 μl of spore suspension (10 6 CFU / mL) of Alternaria alternata, Stemphylium lycopersici and Colletotrichum gloeosporioides was injected, respectively. The inoculated apples were placed in a plastic fresh-keeping box, and the bottom of the fresh-keeping box was covered with wet filter paper to keep the moisture. The fresh-keeping box was placed in a 25°C constant temperature incubator for incubation. After 7 days, the incidence of fruit was investigated. There were 15 apples in each treatment, and the experiment was repeated 3 times.

[0032] From Figure 1It can be seen that the strain GY-2 has obvious inhibitory effect on the lesion area of the black spot disease caused by Alternaria alternata, and the inhibition rate is 90.0%.

[0033] From Figure 2 It can be seen that the strain GY-2 has obvious inhibitory effect on the lesion area of the red rot disease caused by P. rosarum, and the inhibition rate is 88.3%.

[0034] From Figure 3 It can be seen that the strain GY-2 has obvious inhibitory effect on the lesion area of the anthracnose disease caused by C. gloeosporioides, and the inhibition rate is 81.0%.

[0035] Example 3

[0036] Detection of the colonization dynamics of Leuconostoc mesenteroides GY-2 on apple

[0037] The colonization of the strain GY-2 on the apple fruit tissue and the surface of the apple peel was detected respectively. The surface-sterilized apple fruits were used for the inoculation experiment of the strain GY-2. The bacterial suspension (1×10 8 CFU / mL) of the strain GY-2 was uniformly sprayed on the surface of the apple fruits by a small spray bottle until the surface was uniformly attached with the biocontrol bacteria liquid, which was used for detecting the colonization on the peel. 15 μL of the bacterial suspension with the same concentration was inoculated on the surface wound of the apple for detecting the colonization on the fruit tissue. The inoculated apples were placed in a 25℃ incubator, and the colonization was detected at 0, 1, 2, 4, 8, and 16 days. 1 g of the fruit tissue and the peel was collected from the inoculated apple, and was grinded in 10 mL of sterile water. 10 μL of the grinded liquid was uniformly coated on the MRS plate by the dilution plate method, and was placed in a 25℃ incubator for 2 days. The number of colonies on the plate was detected. Each treatment had five replicates, and the experiment was repeated three times.

[0038] The number of colonies of the strain GY-2 on the apple peel and the wound was detected. When the strain GY-2 was inoculated on the fruit for 16 days, the number of colonies of the strain GY-2 on the peel was 1.1×10 5 CFU, and the number of colonies on the wound reached 5.9×10 6 CFU, which indicated that the strain GY-2 successfully colonized on the apple fruit. Figure 4

[0039] Example 4

[0040] Identification of Leuconostoc mesenteroides GY-2.

[0041] ​GY-2 was streaked on MRS medium to observe colony morphology. Gram staining was used to determine whether strain GY-2 was Gram-positive or Gram-negative. Scanning electron microscopy was used to observe the cell morphology of strain GY-2. A phylogenetic tree of strain GY-2 was constructed using 16S rRNA sequence to analyze its taxonomic position.

[0042] like Figure 5 As shown, strain GY-2 appears milky white on MRS solid medium, with a smooth center and raised edges, and is opaque.

[0043] like Figure 6 As shown, Gram staining indicates that the bacterium is Gram-positive.

[0044] like Figure 7 As shown, scanning electron microscopy revealed that strain GY-2 was a short rod-shaped organism without flagella, with a length of 1.4-1.8 μm and a width of 0.6-0.8 μm.

[0045] 16S rRNA gene analysis: DNA was extracted from strain GY-2 using a bacterial DNA extraction kit. PCR amplification of the strain's 16S rRNA gene was performed using universal bacterial primers 27F (5′-AGAGTTTGATCCTGGCTCAG-3′) and 1492R (5′-TACGACTTAACCCCAATCGC-3′). The amplification program was: 94℃ pre-denaturation for 5 min; 35 cycles (94℃ denaturation for 30 s, 56℃ annealing for 30 s, 72℃ extension for 90 s); 72℃ extension for 10 min. PCR products were detected by 1% (w / v) agarose gel electrophoresis, and sequencing was performed by a biotechnology company. Based on the 16S rRNA sequence tree, combined with morphological, Gram staining, and phylogenetic analysis, strain GY-2 was identified as *Leuconostoc mesenteroides* (Enteromorpha repens). Figure 8 ).

[0046] Example 5

[0047] The effects of strain GY-2 on apple fruit quality parameters.

[0048] The influence of the strain GY-2 on the weight (weight loss rate), acidity, soluble solid content and hardness of apple fruits was determined. A number of Fuji apples with uniform size, no surface wounds and uniform color were prepared, and the bacterial suspension of the strain GY-2 was sprayed on the apple fruits with a small spray bottle until the apple surface was fully coated with the bacterial suspension. The control group was sprayed with sterile water. The treated apples were placed in plastic preservation boxes and stored at 25°C for 15 days, after which the four parameters of the fruits were detected. The balance was used to detect the weight change of the fruits, the acidity meter (PAL-Easy / F5, Japan Aotuo) was used to detect the acidity of the fruits, the sugar meter (PAL-1, Japan Aotuo) was used to detect the soluble solid content of the fruits, and the hardness meter (GY-4, China Aidebao) was used to detect the hardness of the fruits.

[0049] As can be seen from Table 1, the strain GY-2 has no obvious influence on the weight, acidity, soluble solid content and hardness of apple fruits.

[0050] Table 1 Influence of the strain GY-2 on the quality parameters of apple fruits

[0051]

Claims

1. A strain of Leuconostoc mesenteroides, GY-2, which was deposited on January 23, 2024, at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC No. 29733.

2. The use of Leuconostoc mesenteroides GY-2 according to claim 1, characterized in that... It can be used to control three types of postharvest fungal diseases in apples and to preserve apples during storage. (1) First, enteromonas mesentery GY-2 was inoculated into MRS culture medium for activation culture to obtain culture medium; culture medium was inoculated at a ratio of 1% into new MRS culture medium for secondary culture; after centrifugation of culture medium, the supernatant was removed, the bacterial cells were collected, and after washing with sterile water, a bacterial suspension was prepared. (2) Select apples with no wounds on the surface, uniform color and size, disinfect them with 2% NaClO for 2 minutes, then rinse them with sterile water, and place them in a plastic preservation box to dry. Puncture wounds of uniform size and depth in the middle of the dried apples, add the bacterial suspension of Leuconostoc mesenteroides GY-2 prepared in step one to each wound, let it stand for 2 hours, and after the bacterial suspension dries, inject spores of Alternaria alternata, Polysporum tinctorium, and Colletotrichum gloeosporioides into the holes respectively. Use apples with an equal amount of water instead of GY-2 bacterial suspension as a control. Place the treated apples in a plastic preservation box and incubate them in an incubator to control postharvest diseases of apples.

3. The method for controlling postharvest diseases of apples using Leuconostoc mesenteroides GY-2 according to claim 2, characterized in that, The MRS culture medium described in step (1) consists of the following per 1L: 10g peptone, 10g beef extract, 5g yeast powder, 20g glucose, 2g diammonium citrate, 5g sodium acetate trihydrate, 2g dipotassium hydrogen phosphate, 0.58g magnesium sulfate heptahydrate, 0.25g manganese sulfate monohydrate, 1ml Tween 80, and distilled water to a final volume of 1000mL. The pH is adjusted to 6.5, and the medium is sterilized at 121℃ for 20min.

4. The method for controlling postharvest diseases of apples using Leuconostoc mesenteroides GY-2 according to claim 2, characterized in that, The conditions for the first and second activation cultures in step (1) are: 28℃ for 15-20h; the conditions for centrifugation are: 25℃, 10000rpm, and 10-15min.

5. The method for controlling postharvest diseases of apples using Leuconostoc mesenteroides GY-2 according to claim 2, characterized in that, The diameter of the hole in step (2) is 3mm and the depth is 3mm.

6. The method for controlling postharvest diseases of apples using Leuconostoc mesenteroides GY-2 according to claim 2, characterized in that, In step (2), the volume ratio of the strain GY-2 suspension to the pathogen suspension added to each wound is 1:1, and both are 15 μl; the concentration of the Leuconostoc mesenteroides GY-2 suspension is 1×108 colony-forming units / mL, and the concentration of the suspensions of Alternaria alternata, Polysporus rosenbergii, and Colletotrichum gloeosporioides is 1×106 spores / mL.

7. The method for controlling postharvest diseases of apples using Leuconostoc mesenteroides GY-2 according to claim 2, characterized in that, In step (2), the temperature for incubation in the constant temperature and humidity incubator is 25°C and the relative humidity is 90%.

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