Salt-tolerant bacillus YH16 and application thereof

Abstract

The invention provides a salt-tolerant bacillus YH16 strain and application thereof, and relates to the technical field of microbial control. The salt-tolerant bacillus YH16 is classified and named as the salt-tolerant bacillus, the salt-tolerant bacillus YH16 is preserved in the China General Microbiological Culture Collection Center on December 2, 2024, and the preservation number is CGMCC No: 32876. The salt-tolerant bacillus YH16 is named as the salt-tolerant bacillus, and the salt-tolerant bacillus YH16 is preserved in the China General Microbiological Culture Collection Center on December 2, 2024. The salt-tolerant bacillus YH16 is applied to prevention and treatment of grapefruit penicilliosis and / or blueberry fruit gray mold. The invention overcomes the defects of the prior art, can effectively control the harm of grapefruit penicilliosis and blueberry fruit gray mold, promotes the improvement of the resistance level of grapefruit and blueberry fruits, and plays a positive role in promoting the sustainable and healthy development of the grapefruit and blueberry planting industry.

Description

Technical Field

[0001] The present invention relates to the technical field of microbial control, and in particular to a halotolerant Bacillus YH16 and an application thereof. Background Art

[0002] The fruit and vegetable industry holds a key position in the global agricultural economy. However, the frequent occurrence of post-harvest diseases poses a significant challenge to the industry. Pathogens such as Penicillium spp. and Botrytis cinerea cause significant losses during the storage, transportation, and sale of fruits and vegetables like grapefruit and blueberries, becoming a significant constraint on the industry's development.

[0003] Penicillium expansum is a widespread fruit spoilage fungus that parasitizes or colonizes the skin and wound tissue of rotting pears, apples, citrus fruits, and other fruits. Its conidia are spread by air currents, insects, or mechanical damage, easily causing widespread infection during fruit storage, transportation, and distribution. Globally, postharvest fruit losses due to Penicillium expansum are as high as 15%-30%, and in temperate fruit-producing regions, the economic losses can reach billions of dollars. The exocellular hydrolases it secretes, such as pectinases and cellulases, rapidly degrade fruit cell walls, causing soft rot. Its metabolite, patulin, is highly cytotoxic and genotoxic. It is stable in acidic environments and can penetrate the peel and deep into the flesh. Residues in juice processing reach 60%-80%, and even pasteurization is difficult to completely remove. Long-term consumption of foods containing patulin can cause acute gastroenteritis, liver and kidney damage, and increase the risk of malignant tumors such as gastric and esophageal cancer. It has been classified as a Group 3 carcinogen by the World Health Organization.

[0004] Gray mold, caused by Botrytis cinerea, is a major airborne disease affecting numerous crops worldwide. It can be widely spread through air, water, and field operations, and is prone to outbreaks in low-temperature, high-humidity environments (20°C-25°C, with relative humidity consistently above 90%). It primarily infects flowers, leaves, and fruits, leading to severe yield reductions of 30%-70%, or even complete crop failure, causing significant economic losses to the greenhouse vegetable and berry industries. For example, blueberry gray mold causes dark brown, V-shaped lesions on leaves, which later develop a gray-black mold layer. Flowers and fruit become water-soaked, soft, and covered in gray-black mold. Grapefruit blue mold develops during storage, initially with water-soaked, light brown, circular lesions. The affected fruit peel softens and rots, then develops white hyphae and transforms into a green mold layer. The fungus also secretes volatile substances that damage the peel of healthy fruit, leading to contact transmission.

[0005] Current prevention and control technologies for these pathogens have significant limitations. In terms of physical control, although low-temperature storage (0-4°C) can inhibit the growth of Penicillium expansum hyphae, it requires a supporting cold chain logistics system, which is costly and can easily cause chilling damage to tropical fruits. Heat treatment (immersion in hot water at 50-55°C) can inactivate spores, but it can easily cause heat damage to the fruit and flavor deterioration. Chemical control mainly relies on fungicides such as benzimidazoles and imidazoles. However, Penicillium expansum has developed resistance to drugs such as carbendazim and thiabendazole, and Botrytis cinerea also has a high risk of resistance. Chemical residues can cause food safety and environmental pollution problems. Technologies such as ozone fumigation and irradiation treatment are difficult to promote and apply in small and medium-sized fruit companies due to high equipment costs and complex operations.

[0006] Against this backdrop, biocontrol technologies, owing to their environmental, safety, and sustainability advantages, have shown significant potential and become a research hotspot. Antagonistic microorganisms such as yeasts and Bacillus can inhibit pathogen growth through mechanisms such as nutrient competition, secretion of antimicrobial peptides, and induction of fruit resistance. Some yeast strains can reduce the incidence of blue mold in apples by 70%-90%. Bacillus amyloliquefaciens JK-1, isolated from blueberry leaves, has an inhibitory rate of over 81% against Botrytis cinerea. It secretes antimicrobial substances that cause mycelial expansion, plasmolysis, and cell membrane rupture in the pathogen, and can also modulate pathogen genes to influence its growth. However, in practice, conventional Bacillus strains are inhibited in saline soils, high-salt processing environments (such as in some fruit and vegetable pickling processes), or salt-stressed storage, limiting their effectiveness in these specialized habitats. Summary of the Invention

[0007] In response to the shortcomings of the existing technology, the present invention provides a salt-tolerant Bacillus YH16 and its application. The YH16 strain can effectively control the damage caused by blue mold disease of grapefruit fruit and gray mold disease of blueberry fruit, while promoting the improvement of the resistance level of grapefruit fruit and blueberry fruit, and has a positive promoting effect on the sustainable and healthy development of the grapefruit and blueberry planting industries.

[0008] To achieve the above objectives, the technical solution of the present invention is implemented through the following technical solutions: In the first aspect, the present invention provides a salt-tolerant Bacillus ( Bacillus halotolerans ) YH16, the classification of the halodurable Bacillus YH16 is named as halodurable Bacillus Bacillus halotolerans , and was deposited in the General Microbiology Center of China Culture Collection Administration on December 2, 2024, with the deposit number CGMCC No: 32876.

[0009] In a second aspect, the present invention provides a halotolerant Bacillus agent comprising the halotolerant Bacillus strain and its metabolites.

[0010] In a third aspect, the present invention proposes the use of the halotolerant Bacillus strain or the halotolerant Bacillus agent in the prevention and treatment of post-harvest diseases of fruits and vegetables.

[0011] Furthermore, the post-harvest diseases of fruits and vegetables are caused by Penicillium expansum or Botrytis cinerea, and the application method is to prepare the strain or microbial agent into a concentration of 10 6 -10 8 Fruits and vegetables were treated with bacterial solution containing 100mg / mL.

[0012] The fruits and vegetables include at least one of grapefruit, blueberry, pome, apple, and citrus.

[0013] Preferably, the grapefruit Penicillium pathogenic fungus is Penicillium italicum ( Penicillium italicum ), Penicillium digitatum ( Penicillium digitatum )、Penicillium expansum( Penicillium expansu ) any one of the following; the blueberry gray mold pathogen is Botrytis cinerea ( Botrytis cinerea ).

[0014] The present invention provides a halotolerant Bacillus subtilis YH16 and its application, which have the following advantages over the prior art: The present invention provides a salt-tolerant Bacillus ( Bacillus halotolerans ) YH16 strain, which is effective against grapefruit pathogen Penicillium expansum Penicillium expansu The inhibition rate reached 59.96% against the pathogenic fungus of blueberry gray mold. Botrytis cinerea The inhibition rate reached 86%. Soaking grapefruit and blueberry fruits in a spore suspension of Haloperidol Bacillus YH16 achieved a 32.5% control effect against the pathogenic fungus P. expansu. On the 12th day, the diameter of the lesions on the blueberry fruits was 6.83 mm. While the entire control group was diseased (100% incidence), the incidence after inoculation with Haloperidol Bacillus YH16 was only 56.6%, effectively controlling blue mold on grapefruit and gray mold on blueberries. BRIEF DESCRIPTION OF THE DRAWINGS

[0015] Figure 1 Figure A shows the colony morphology of strain YH16 on LB medium in Example 1 of the present invention; Figure B shows the Gram staining and spore staining results of strain YH16; Figure 2 The growth results of the plate confrontation test in Example 2 of the present invention after 5 days of inoculation are shown, where A is the control group and B is the treatment group (YH16); Figure 3 This is a bar graph showing the inhibition rate of the treated group (YH16) against Penicillium expansum colonies at the same time in Example 2 of the present invention; Figure 4The growth results of the plate confrontation test in Example 3 of the present invention after 2 days of inoculation are shown, where A is the control group and B is the YH16 group; Figure 5 This is a bar graph showing the inhibition rate of the treatment group (YH16) against Botrytis cinerea colonies at different treatment times in Example 3 of the present invention; Figure 6 This is a schematic diagram comparing the decay conditions of the control group (A) and the treatment group (B) in the grapefruit Penicillium expansum control experiment 10 days after Example 4 of the present invention; Figure 7 This is a bar graph showing the comparison of lesion diameters of the control group (CK) and the treatment group (YH16) at different treatment times in the experiment on the control effect of Penicillium expansum on grapefruit in Example 4 of the present invention; Figure 8 This is a bar graph showing the control effect of the treatment group (YH16) at different treatment times in the grapefruit Penicillium expansum control effect experiment in Example 4 of the present invention; Figure 9 This is a schematic diagram of the disease conditions of blueberries in the control group (A) and the YH16 group (B) at 8 days of the experiment on the control effect of Botrytis cinerea on blueberries in Example 5 of the present invention; Figure 10 This is a bar graph comparing the lesion diameters of the control group (CK) and the treatment group (YH16) at different treatment times in the experiment on the control effect of blueberry botrytis cinerea in Example 5 of the present invention; Figure 11 This is a bar graph showing the incidence rates of the control group (CK) and the treatment group (YH16) at different treatment times in the blueberry Botrytis cinerea control effect experiment in Example 5 of the present invention. DETAILED DESCRIPTION

[0016] To make the objectives, technical solutions, and advantages of the embodiments of the present invention more clear, the technical solutions in the embodiments of the present invention are clearly and completely described below in conjunction with the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. All other embodiments obtained by ordinary technicians in this field based on the embodiments of the present invention without making any creative efforts shall fall within the scope of protection of the present invention.

[0017] The following halotolerant Bacillus subtilis YH16 strain was isolated from the roots of healthy grapefruit plants in Xishuangbanna, Yunnan Province. It has strong extracellular hydrolase activities, including chitinase, cellulase, β-1,3-glucanase, and protease. It is deposited in the General Microbiology Center of the China Culture Collection Administration, at No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, and is classified as Halotolerant Bacillus subtilis Bacillus halotolerans , strain number is YH16, preservation time is: December 2, 2024, preservation number is: CGMCC No: 32876. Example 1:

[0018] Identification of the species of halotolerant Bacillus subtilis YH16: 1. Morphological identification LB medium formula: peptone 10 g, yeast powder 5 g, sodium chloride 10 g, agar powder 15 g, dilute to 1000 mL with distilled water, sterilize at 121 °C for 20 min, and set aside.

[0019] Inoculate the halodurable Bacillus strain YH16 onto an LB plate and invert the plate at 28°C for 24 hours. Record and describe the colony morphology, color, and other characteristics. Select a small number of cells for Gram staining and observe the staining and bacterial characteristics using an optical microscope.

[0020] like Figure 1 As shown, the strain forms yellowish opaque colonies with a rough, raised surface, irregular edges, and a certain thickness in LB solid medium ( Figure 1 In the figure (A), the Gram staining showed purple, indicating that the strain was a Gram-positive bacterium, and the spore staining results showed that the strain could produce spores, indicating that it had the ability to produce spores ( Figure 1 In middle B, the bacteria are blue, the spores are red, and the yellow arrows point to the spores. Bar = 20 μm).

[0021] 2. Molecular Biology Identification DNA of Bacillus halodurans YH16 was extracted using a bacterial DNA extraction kit (Cat: YZ-D3350, Beijing Solebeau Technology Co., Ltd.). 16S rDNA universal primers were used for PCR amplification. The amplified products were detected by gel electrophoresis and sent to Kunming Shuoqing Biotechnology Co., Ltd. for bidirectional sequencing. The sequencing results were compared with sequences of similar model strains using the NCBI website (https: / / www.ncbi.nlm.nih.gov). A phylogenetic tree of the 16S rDNA gene sequences was constructed using the Newton-Jones method using MEGA11 software.

[0022] The 16SrDNA sequence of the halodurable Bacillus subtilis YH16 is shown in SEQ ID NO.1: SEQ ID NO.1: Example 2:

[0023] The plate confrontation culture was used to determine the effect of halotolerant Bacillus sp. YH16 on the pathogenic fungus Penicillium expansum of grapefruit penicillium disease. Penicillium expansu ) inhibition results: 10 μL of the purified Penicillium expansum spore suspension was inoculated on the filter paper in the center of the PDA culture dish. Four sterile filter paper discs were placed 3 cm away from the center. 10 μL of the fermentation suspension of Bacillus halophilus YH16 (concentration of 1×10 7 Each treatment was repeated three times independently. The colony diameters were counted after culturing at 28°C in the dark for 5 days, and the inhibition rate was calculated.

[0024] Penicillium expansum Penicillium expansu The antagonistic ability of the fungus was determined by the cross-cross method, and the calculation formula was: R(%) = (R1−R2) / R1×100%, where R1 represented the colony diameter of the pathogen in the control, and R2 represented the colony diameter of the pathogen in the antagonism test.

[0025] Specific results can be found in Figure 2 and Figure 3 As shown, it can be seen that Figure 2 Middle B is the plate of halotolerant Bacillus YH16 10 days after inoculation, with obvious inhibition zone appearing. Figure 2 A is a plate treated with sterile water control group, with no inhibition zone, indicating that halodurable Bacillus YH16 can significantly inhibit the growth of Penicillium expansum, and according to Figure 3 It can be seen that the inhibition rate gradually increased from the 5th day, and reached 61.35% on the 10th day. Example 3:

[0026] Plate confrontation culture was used to determine the effect of salt-tolerant Bacillus YH16 on the blueberry gray mold pathogen Botrytis cinerea ( Botrytis cinerea ) inhibition results: 10 μL of the purified Botrytis cinerea spore suspension was inoculated on the filter paper in the center of the PDA culture dish. Four sterile filter papers were placed 3 cm away from the center. 10 μL of the fermentation suspension of Bacillus halophilus YH16 (concentration of 1×10 7 Each treatment was repeated three times independently, and the colony diameters were counted after culturing at 28°C in the dark for 2 days.

[0027] The antagonistic ability to Botrytis cinerea was observed and determined, and the colony diameter was determined by the cross method.

[0028] Specific results such as Figure 4 and Figure 5 As shown, Figure 4 B in the middle shows that after 2 days of inoculation, the salt-tolerant Bacillus YH16 showed an obvious inhibition zone and could significantly inhibit the growth of Botrytis cinerea. Figure 4 A is the plate confrontation of the experimental control group, with no inhibition zone; Figure 5 It can be seen that the inhibition rate gradually increased from the 4th day, and reached 86% on the 8th day. Example 4:

[0029] Effect of spore suspension of Bacillus halodurans YH16 on the growth of Penicillium expansum in grapefruit ( Penicillium expansu )’s control effect: 1. Fruit Selection: Grapefruit fruits of the Rio Red variety were purchased from the Donghua Farmers' Market in Kunming, Yunnan Province. Undamaged fruits of uniform size, firmness, and maturity, uniform color and gloss, and luster were selected for testing. The surfaces of the test samples were pre-washed with tap water, disinfected with 75% alcohol, and then dried at room temperature.

[0030] 2. Preparation of test bacteria: Penicillium expansum ( Penicillium expansum ) strains were stored in the laboratory. The pathogenic strain was activated by culturing on potato dextrose agar (PDA) at 28°C for 7 days. The pathogen spores on the plate were first rinsed with sterile distilled water and transferred to a sterile centrifuge tube (filled with 2 mL of sterile water). After thorough shaking, the spore concentration was adjusted to 1×10 using a hemocytometer. 5 The halodurable Bacillus YH16 used in the test should be activated at least twice in LB liquid medium before use, and a suspension of Penicillium expansum spores should be prepared. The concentration should be adjusted to 1×10 spores / mL using a hemocytometer before use. 7 / mL, set aside.

[0031] 3. The experiment consisted of treatment and control groups. In the treatment group, evenly spaced holes (approximately 5 mm deep and 4 mm wide) were made at the equator of the fruit using a sterile pipette tip. Each hole was inoculated with 20 μL of a spore suspension of Bacillus halodurans YH16. In the control group, each hole was inoculated with an equal volume of sterile water. Three hours later, 20 μL of a spore suspension of Penicillium expansum was injected into each wound. After inoculation, the fruit was placed in packaging at room temperature. After five days, the fruit wounds were observed for infection, and the diameter of the lesions was recorded. Lesion diameters greater than 15 mm were determined using the cross-hatch method. Nine fruits were included in each treatment, for a total of 18 fruits in two groups, with two replicates. The control efficacy was calculated as (lesion diameter in control group - lesion diameter in treatment group) / lesion diameter in control group × 100%.

[0032] The results are as follows Figure 6 As shown in the figure, 10 days after inoculation with the spore suspension of Bacillus halophilus YH16 and pathogens, it was found that the spore suspension of Bacillus halophilus YH16 significantly inhibited the expansion of the rot diameter of grapefruit fruit ( Figure 6 (B) effectively controlled the postharvest rot caused by Penicillium expansum in the fruit wounds, while the control group showed no significant improvement ( Figure 6 (A). And if Figure 7 and Figure 8 As shown in the figure, pre-inoculation with spore suspension of Bacillus halophilus YH16 significantly reduced the lesion diameter of grapefruit fruit. On the 10th day, the lesion diameter was 2.8 cm, and the control effect reached 32.5%. Example 5:

[0033] Effect of spore suspension of salt-tolerant Bacillus sp. YH16 on blueberry gray mold (Botrytis cinerea) Botrytis cinerea )’s control effect: 1. Fruit Selection: Blueberries of the "Jewel" variety were purchased from the Donghua Farmers' Market in Kunming, Yunnan Province. Undamaged blueberries of uniform size, firmness, and maturity, uniform color and gloss, and undamaged were selected for testing. The surface of the test samples was pre-washed with tap water, disinfected with 75% alcohol, and then dried at room temperature.

[0034] 2. Preparation of test bacterial solution: Botrytis cinerea isolated from diseased blueberry fruits ( Botrytis cinerea ) strains were stored in the laboratory. The pathogenic strain was activated by culturing on potato dextrose agar (PDA) at 28°C for 7 days. The pathogen spores on the plate were first rinsed with sterile distilled water and transferred to a sterile centrifuge tube (filled with 2 mL of sterile water). After thorough shaking, the spore concentration was adjusted to 1×10 using a hemocytometer. 5 The halodurable Bacillus YH16 used in the test should be activated at least twice in LB liquid medium before use, and a spore suspension should be prepared. The concentration should be adjusted to 1×10 using a hemocytometer before use. 7 / mL, set aside.

[0035] 3. Soak the blueberry fruits in 2% sodium hypochlorite for 1 minute for disinfection. After natural drying, the fruits were divided into 2 groups, with 9 fruits in each group, for a total of 18 fruits. Use a sterilized punch to punch a hole with a diameter and depth of 4 mm at the equator of the blueberry fruit. After the juice in the hole has drained, 5 μL of the treatment solution of Bacillus halophilus YH16 was quantitatively injected into each hole of the fruit. The control group (CK) was sterile water. After 2 hours, 5 μL of the gray mold spore suspension (concentration of 1×10 5CFU / mL), after all the treated liquid was absorbed, the individual fruits were packaged in self-sealing bags and stored in plastic baskets at room temperature. Nine fruits were used for each treatment. After inoculation, the fruits were placed in a packaging box at room temperature. After 5 days, the fruit wound infection was observed and the lesion diameter was recorded. When the diameter was greater than 5 mm, the lesion diameter was determined by the cross-cross method. Nine fruits were used for each treatment, 18 fruits in total in 2 groups, and repeated twice. Prevention and control effect = (lesion diameter of control group - lesion diameter of treatment group) / lesion diameter of control group × 100%. Calculation of inoculation incidence rate (%) = total number of diseased holes in the fruit / total number of inoculated holes in the fruit × 100%.

[0036] The incidence of blueberry fruit was investigated and counted. Figures 9-11 As shown in the figure, it can be seen that the inoculation of the spore suspension of Bacillus halophilus YH16 significantly inhibited the expansion of the rot diameter of blueberry fruit and the increase in the incidence of fruit, and effectively controlled the growth of Botrytis cinerea in the fruit wound. Botrytis cinerea Postharvest decay caused by Figure 9 , the left side is the control group, the right side is the treatment group). Among them, pre-inoculation with halotolerant Bacillus YH16 significantly reduced the diameter of the lesions on blueberry fruits. On the 12th day, the diameter of the lesions was 6.83 cm ( Figure 10 When all the control group (CK) became diseased (storage day 8, disease incidence rate 100%), the disease incidence rate after inoculation with halotolerant Bacillus sp. YH16 was 56.6% ( Figure 11 ).

[0037] The pathogenic fungus of grapefruit Penicillium is Penicillium italicum ( Penicillium italicum ), Penicillium digitatum ( Penicillium digitatum )、Penicillium expansum( Penicillium expansu ) any one of the following; the blueberry gray mold pathogen is Botrytis cinerea ( Botrytis cinerea ).

[0038] It should be noted that, in this document, relational terms such as first and second, etc., are used only to distinguish one entity or operation from another entity or operation, and do not necessarily require or imply the existence of any such actual relationship or order between these entities or operations. Moreover, the terms "comprises," "comprising," or any other variants thereof are intended to cover non-exclusive inclusion, so that a process, method, article, or device comprising a series of elements includes not only those elements, but also other elements not explicitly listed, or elements inherent to such process, method, article, or device. In the absence of further limitations, an element defined by the phrase "comprising a ..." does not exclude the presence of other identical elements in the process, method, article, or device comprising the element.

[0039] The above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit the same. Although the present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art should understand that they can still modify the technical solutions described in the aforementioned embodiments, or make equivalent replacements for some of the technical features therein. However, these modifications or replacements do not deviate the essence of the corresponding technical solutions from the spirit and scope of the technical solutions of the various embodiments of the present invention.

Claims

1. A strain of halotolerant Bacillus ( Bacillus halotolerans ) YH16, characterized by: The classification of the halogen-tolerant Bacillus subtilis YH16 is named as halogen-tolerant Bacillus subtilis Bacillus halotolerans , and was deposited in the General Microbiology Center of China Culture Collection Administration on December 2, 2024, with the deposit number CGMCC No: 32876.

2. A salt-tolerant Bacillus agent, characterized in that The invention comprises the halodurable Bacillus strain according to claim 1 and its metabolites.

3. Use of the halogen-tolerant Bacillus strain according to claim 1 or the halogen-tolerant Bacillus agent according to claim 2 in the prevention and control of postharvest diseases of fruits and vegetables.

4. The use according to claim 3, characterized in that The post-harvest diseases of fruits and vegetables are caused by Penicillium expansum or Botrytis cinerea. The application method is to prepare the strain or microbial agent to a concentration of 10 6 -10 8 Fruits and vegetables were treated with bacterial solution containing 100mg / mL.

5. The use according to claim 4, characterized in that The fruits and vegetables include at least one of grapefruit, blueberry, pome, apple, and citrus.

6. The use according to any one of claims 3 to 5, characterized in that: The grapefruit Penicillium pathogenic fungus is Penicillium italicum ( Penicillium italicum ), Penicillium digitatum ( Penicillium digitatum )、Penicillium expansum( Penicillium expansu ) any one of the following; the blueberry gray mold pathogen is Botrytis cinerea ( Botrytis cinerea ).

7. Use of the halodurable Bacillus strain according to claim 1 or the halodurable Bacillus agent according to claim 2 in treating grapefruit penicillium mold and / or blueberry gray mold.

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