Pantoea rodasi YBZ4 for preventing and treating leaf scorch of walnuts and application of Pantoea rodasi YBZ4

Abstract

The invention relates to the technical field of microorganisms, in particular to Pantoea rodasi YBZ4 for preventing and treating the leaf scorch disease of walnuts and application of the Pantoea rodasi YBZ4. The Pantoea rodasi YBZ4 for preventing and treating the walnut leaf scorch disease is preserved in the China General Microbiological Culture Collection Center (CGMCC), and the preservation number of the Pantoea rodasi YBZ4 is CGMCC No.31371. The Pantoea rodasi YBZ4 can effectively relieve the occurrence of leaf scorch of walnut seedlings, the damage degree of leaves of the walnut seedlings under salt stress is remarkably reduced, and the prevention and treatment effect can reach 90% or above.

Description

Technical Field

[0001] The invention relates to the technical field of microorganisms, and in particular to a Pantoearodasii YBZ4 for preventing and treating walnut leaf scorch disease and an application thereof. Background Art

[0002] Walnut (Juglans regia L.) is a plant of the genus Juglans in the family Juglandaceae. It is distributed in Northwest, Southwest, North China and South my country. Walnut leaf necrosis (JLN) is a physiological disease. The disease starts from the edge of the walnut leaves and then extends to the center, gradually spreading to the entire leaf. The walnut plants with the disease show brown and scorched leaf edges, hindered photosynthesis, and black and unfull fruits, which seriously affect the yield and quality of walnuts. The disease was first reported in the main walnut producing areas in southern Xinjiang in 2012. In recent years, the disease area has increased year by year, the time of occurrence has been earlier, and the degree of occurrence has become more serious. Walnut leaf necrosis may be caused by high temperature and dry hot winds that cause strong plant transpiration, transpiration consumption is greater than absorption, and the surface water evaporates rapidly, rainfall is small, and irrigation is insufficient, causing leaf scorch; it may also be due to the presence of a large amount of Na in salinized soil. + and Cl - , resulting in the accumulation of a large amount of toxic ions in the walnut leaves, breaking the ion homeostasis and inducing ion poisoning, which damages the leaf function and causes leaf scorch. In addition, factors such as weak tree vigor, soil compaction, or inadequate salt and alkali removal measures are also likely to cause walnut leaf scorch. The occurrence of walnut leaf scorch has a serious impact on the yield and quality of the fruit. Some orchards have reduced production by more than 60%. When the disease is severe, it may even cause the tree to become weak and die, seriously threatening the healthy development of the Xinjiang walnut industry. Therefore, it is urgent to develop practical prevention and control measures and carry out effective prevention and control of walnut leaf scorch.

[0003] Traditional improvement methods mainly include building drainage channels, adopting surface irrigation, leaching soil salinity, lowering groundwater levels, improving soil structure, strengthening plant pruning, and improving light conditions. However, these prevention and control measures consume manpower and material resources and are slow to take effect, and cannot effectively solve the problem. In recent years, spraying anti-tidal agents to reduce walnut water loss, applying organic fertilizers such as humic acid and potassium humate, and increasing soil organic matter content can effectively prevent and control the occurrence of walnut leaf scorch, but the economic cost is relatively high. Beneficial bacteria in the rhizosphere of plants have the functions of dissolving phosphorus, potassium, and fixing nitrogen. They can also secrete IAA, ACC deaminase, and produce iron carriers and other substances. They can alleviate the abiotic stress of plants by regulating plant ion homeostasis and reducing oxidative damage. It is an important way to achieve green prevention and control of walnut leaf scorch. The development and application of beneficial microbial preparations for alleviating walnut leaf scorch by exploring beneficial rhizosphere bacteria that can alleviate walnut leaf scorch has attracted widespread attention.

[0004] In view of this, the present invention is proposed. Summary of the invention

[0005] The invention aims to provide a Pantoea rodasii YBZ4 for preventing and treating walnut leaf scorch disease and an application thereof. The Pantoea rodasii YBZ4 is separated from rhizosphere soil of walnut, can alleviate the damage to walnut caused by salt stress, and has the effect of preventing and treating walnut leaf scorch disease.

[0006] In order to achieve the above-mentioned purpose of the present invention, the following technical solutions are particularly adopted:

[0007] One aspect of the present invention relates to a strain Pantoea rodasii YBZ4 for preventing and treating walnut leaf scorch disease, which is deposited in the General Microbiological Center of China Microbiological Culture Collection Administration with a deposit number of CGMCC No.31371.

[0008] The Pantoea rodasii YBZ4 is isolated from walnut rhizosphere soil, can improve the growth ability of walnut under salt stress, has the characteristics of rapid colonization in the walnut root system, has a good prevention and control effect on walnut leaf scorch disease, and has good application prospects.

[0009] Another aspect of the present invention also relates to a microbial preparation, which comprises the Pantoea rodasii YBZ4 described in claim 1.

[0010] The microbial preparation, whose effective ingredient includes Pantoea rodasii YBZ4, belongs to a plant beneficial microbial preparation and can be used to reduce the occurrence of walnut leaf scorch disease. The microbial preparation has good control effect on walnut leaf scorch disease, has a short onset time after application, reduces the control cost of walnut leaf scorch disease, and is environmentally friendly. The microbial preparation can also be used in combination with physical control and chemical control to further improve the control effect of walnut leaf scorch disease.

[0011] Furthermore, the microbial preparation includes the cells of Pantoea rodasii YBZ4 and / or its bacterial suspension. According to the verification of the present invention, the cells of Pantoea rodasii YBZ4 and its bacterial suspension have good prevention and treatment effects on walnut leaf scorch.

[0012] The present invention does not specifically limit the dosage form of the microbial preparation, and conventional dosage forms in the art can be applied to the technical solution of the present invention. In some specific embodiments, the microbial preparation includes but is not limited to: a solid preparation or a liquid preparation.

[0013] Furthermore, when the microbial preparation is a solid preparation, the number of viable bacteria of Pantoearodasii YBZ4 contained in the microbial preparation is 1.19×10 8 ~1.58×10 8 CFU / g.

[0014] Furthermore, when the microbial preparation is a liquid preparation, the number of viable bacteria of Pantoearodasii YBZ4 contained in the microbial preparation is 1.19×10 8 ~1.58×10 8 CFU / mL.

[0015] Limiting the number of live bacteria of Pantoea rodasii YBZ4 in the microbial preparation within a certain range can ensure that the microbial preparation can exert the best prevention and treatment effect of walnut leaf scorch disease and provide theoretical dosage guidance for the practical application of Pantoea rodasii YBZ4.

[0016] Furthermore, the microbial preparation also includes conventional auxiliary materials in this field.

[0017] Another aspect of the present invention also relates to a method for preventing and treating walnut leaf scorch, comprising applying the Pantoea rodasii YBZ4 and / or the microbial preparation to walnuts.

[0018] The present invention does not specifically limit the application method, and any conventional application method in the art can be used to implement the technical solution of the present invention. In some specific embodiments, the application method includes but is not limited to: root irrigation.

[0019] Another aspect of the present invention also relates to the use of the Pantoea rodasii YBZ4 or the microbial preparation in preventing and controlling walnut diseases.

[0020] Furthermore, the walnut diseases include: diseases caused by salt stress.

[0021] Furthermore, the walnut diseases include: walnut leaf scorch.

[0022] Deposit information of strain YBZ4: Pantoena rodasii YBZ4 was deposited in the General Microbiology Center of China Microorganism Culture Collection Administration on July 19, 2024, with the deposit number CGMCC No. 31371. Deposit address: Institute of Microbiology, Chinese Academy of Sciences, No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, China, Postal Code 100101.

[0023] Compared with the prior art, the present invention has the following beneficial effects:

[0024] (1) The Pantoea rodasii YBZ4 provided by the present invention is isolated from the rhizosphere soil of walnut, can be colonized in walnut plants, can effectively reduce the occurrence of walnut seedling leaf scorch, and significantly reduce the degree of damage to the leaves of walnut seedlings under salt stress, with a control effect of more than 90%.

[0025] (2) The microbial preparation provided by the present invention comprises Pantoea rodasii YBZ4 as an active ingredient, and is a plant beneficial microbial preparation, which can be used to improve the growth ability of walnuts under salt stress. The microbial preparation has good control effect on walnut leaf scorch disease, has a short onset time after application, reduces the control cost of walnut leaf scorch disease, and is environmentally friendly. The microbial preparation can also be used in combination with physical control and chemical control to further improve the control effect of walnut leaf scorch disease. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] In order to more clearly illustrate the specific implementation methods of the present invention or the technical solutions in the prior art, the drawings required for use in the specific implementation methods or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are some implementation methods of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0027] Figure 1 shows the colony morphology of strain YBZ4 (scale bar: 2 mm);

[0028] Figure 2 The effect of strain YBZ4 on the malondialdehyde content in walnut seedlings with scorched leaf disease;

[0029] Figure 3 The effect of strain YBZ4 on the catalase content in walnut seedlings with scorched leaf disease;

[0030] Figure 4 The effect of strain YBZ4 on the chlorophyll content of walnut seedlings with scorched leaf disease (A is the chlorophyll a content, B is the chlorophyll b content, and C is the total chlorophyll content);

[0031] Figure 5 The effect of strain YBZ4 on the proline content in walnut seedlings with scorched leaf syndrome;

[0032] Figure 6 The effects of strain YBZ4 on the alleviation of scorched leaf symptoms in walnut seedlings (A is the water control group, B is the NaCl treatment group, and C is the strain YBZ4+NaCl treatment group, scale bar: 5 cm);

[0033] Figure 7 The strain YBZ4 alleviated the severity of leaf damage in walnut seedlings;

[0034] Figure 8 This is the control effect of strain YBZ4 on walnut leaf scorch. DETAILED DESCRIPTION

[0035] The technical scheme of the present invention will be clearly and completely described below in conjunction with the accompanying drawings and specific embodiments, but it will be understood by those skilled in the art that the following described embodiments are part of embodiments of the present invention, rather than all embodiments, and are only used to illustrate the present invention, and should not be considered as limiting the scope of the present invention. Based on the embodiments in the present invention, all other embodiments obtained by those of ordinary skill in the art without making creative work are within the scope of protection of the present invention. If specific conditions are not specified in the embodiments, they are carried out according to normal conditions or conditions recommended by the manufacturer. If the manufacturer is not specified in the reagents or instruments used, they are all conventional products that can be purchased commercially.

[0036] Example 1 Molecular biological identification and colony morphology of strain YBZ4

[0037] (1) 16S rRNA gene sequencing analysis of strain YBZ4

[0038] The genomic DNA of strain YBZ4 was extracted and used as a template to perform PCR amplification using universal primers 27F: 5'-AGAGTTTGATCMTGGCTCAG-3' and 1492R: 5'-TACGGYTACCTTGTTACG ACTT-3' according to the following reaction system (Table 1).

[0039] Table 1 PCR reaction system

[0040]

[0041]

[0042] After the PCR products were checked for correctness by 1% agarose gel electrophoresis, the amplified products with the correct band positions in the electrophoresis results were sent to Ruiboxingke Biotechnology Co., Ltd. for sequencing.

[0043] The 16S rRNA gene sequencing results of strain YBZ4 are shown in SEQ ID No. 1 below:

[0044] SEQ ID No.1:

[0045]

[0046] The obtained sequence was searched by BLAST in the National Center for Bioinformation (NCBI) of the United States. The highest similarity with the 16SrRNA gene fragment sequence of strain YBZ was Pantoea rodasii, with a similarity of 99.78%. Therefore, strain YBZ4 was identified as Pantoea rodasii and named Pantoea rodasii YBZ4. Strain YBZ4 was deposited in the General Microbiology Center of China Microbiological Culture Collection Administration with a deposit number of CGMCC No.31371 and a deposit date of July 19, 2024.

[0047] (2) Colony morphology of strain YBZ4

[0048] Strain YBZ4 was inoculated onto LB solid medium and cultured in a 30°C constant temperature incubator for 24 h. It was observed that the colonies were light yellow, the center of the colonies was bulging, and the surface and edges of the colonies were smooth and moist (see Figure 1 ).

[0049] (3) Physiological and biochemical properties of strain YBZ4

[0050] The detection of physiological and biochemical characteristics of strain YBZ4 mainly refers to the "Handbook of Identification of Common Bacteria Systems", and the culture medium is prepared to detect the ability of strain YBZ4 to secrete methyl red, amylase, protease, lipase, catalase, hydrogen peroxide and produce IAA. No hydrolysis circle was observed in the protein solid culture medium and fat culture medium for strain YBZ4, indicating that the strain cannot secrete protease and lipase, and cannot hydrolyze protein and fat; hydrolysis circle was observed in the starch culture medium for strain YBZ 4, indicating that the strain has the ability to hydrolyze starch. The methyl red reaction of strain YBZ 4 was negative, and the test results of hydrogen peroxide, catalase and IAA were positive. The results of the physiological and biochemical character detection of strain YBZ4 are shown in Table 2:

[0051] Table 2 Physiological and biochemical characteristics of strain YBZ4

[0052]

[0053] +: The presence of a hydrolysis circle or a color reaction indicates that the bacteria can secrete the substance; -: No hydrolysis circle or color reaction appears, indicating that the bacteria cannot secrete the substance.

[0054] Example 2 Preparation of bacterial suspension of strain YBZ4

[0055] The strain YBZ4 preserved in Example 2 was activated on solid LB medium and cultured until a single colony was produced. The single colony was picked and added to 5 mL of liquid LB medium and cultured at 200 rpm and 30°C overnight. The next day, all the bacterial solution was transferred to a triangular flask with 200 mL of LB liquid medium added and cultured at 200 rpm and 30°C overnight. Then, the whole bacterial solution was centrifuged at 6000 rpm for 10 min, and the supernatant was discarded to retain the bacterial precipitate. The bacterial precipitate was resuspended with sterile water and the concentration of the bacterial solution was adjusted to finally obtain 200 mL of a final concentration of 1.19×10 9 ~1.58×10 9 CFU / mL of bacterial suspension was diluted to a final concentration of 1.19×10 8 ~1.58×10 8 CFU / mL of 1L bacterial suspension.

[0056] Example 3: Experiment on salt tolerance and growth promotion of walnut seedlings in greenhouse

[0057] After the walnut seedlings germinate, water them once every 4 days, 80 mL per pot of walnut seedlings, to maintain appropriate humidity in the pot. About 15 days after the seedlings germinate, carry out the salt-tolerant bacteria inoculation test. The standards for the test seedlings are as follows:

[0058] Seedling height>15cm and total number of leaves>10.

[0059] The bacterial inoculation method is to obtain a bacterial suspension of strain YBZ4 by the method of Example 2, select seedlings that germinate and grow to a plant height of>15cm and a total number of leaves>10, and inoculate strain YBZ4 once every 4 days by root irrigation. Each pot is irrigated with 80mL of bacterial suspension, and the bacteria are inoculated 3 times in total; the NaCl treatment group and the clear water control group need to be watered with an equal amount of clear water simultaneously. Salt treatment can be started after the inoculation is completed. The implementation method is that the NaCl control group and the strain YBZ4+NaCl treatment group are watered with 80mL of 75mM NaCl solution every 4 days, and the clear water control group is simultaneously watered with an equal amount of clear water, for a total of 6 times.

[0060] Example 4 Determination of malondialdehyde content

[0061] Malondialdehyde is a product of cell membrane lipid peroxidation, and its production will aggravate the damage to the cell membrane. In this experiment, a malondialdehyde content kit (Suzhou Grace Biotechnology Co., Ltd.) was used to detect the malondialdehyde content in the leaves of walnut seedlings with different treatments.

[0062] The results showed that the MDA content of walnut seedlings in the NaCl treatment group was 24.93 nmoL / g, while that in the strain YBZ4+NaCl treatment group was only 12.65 nmoL / g, which was 49.26% lower than that in the NaCl treatment group; although it was slightly higher than that in the water control group, the difference was not significant, indicating that the inoculated strain YBZ4 could significantly reduce the membrane damage of walnut seedlings under salt stress (see Figure 2 ).

[0063] Example 5 Determination of peroxidase content

[0064] The activity of catalase can evaluate the degree of reactive oxygen toxicity to plants. The higher the catalase content, the less toxicity the plants suffer under stress conditions. In this experiment, a peroxidase content kit (Suzhou Grace Biotechnology Co., Ltd.) was used to detect the peroxidase content in walnut leaves with different treatments.

[0065] The results showed that the peroxidase content of walnut seedlings in the NaCl treatment group was 44.35 μmoL / min / g. After inoculation with strain YBZ4 under salt stress, the peroxidase content of walnut seedlings reached 94.55 μmoL / min / g, which was significantly increased by 113.19% compared with the NaCl treatment group. This showed that inoculation with strain YBZ4 could effectively alleviate the toxicity of active oxygen in walnut seedlings (see Figure 3 ).

[0066] Example 6 Determination of chlorophyll content

[0067] Take walnut leaves with different treatments, rinse them quickly with sterile water and quickly dry them with filter paper, weigh 0.3g of each sample, and repeat 3 times for each treatment. Put 0.3g of sample into a pre-cooled mortar, add an appropriate amount of 95% ethanol solution and quickly crush it into a homogenate; transfer the homogenate to a 50mL centrifuge tube, add 5mL of 95% ethanol solution to wash the precipitate, repeat 2 times; make the volume to 25mL, centrifuge at 4℃ and 9000rpm for 10min; filter, take the supernatant and measure the absorbance at 645nm and 663nm respectively.

[0068] The results showed that the contents of chlorophyll a (Chla), chlorophyll b (Chlb) and total chlorophyll in walnut seedlings in the NaCl treatment group were 0.93 mg / g, 0.43 mg / g and 1.37 mg / g, respectively. After inoculation with strain YBZ, the contents of chlorophyll a (Chla), chlorophyll b (Chlb) and total chlorophyll in walnut seedlings were 1.52 mg / g, 0.83 mg / g and 2.36 mg / g, respectively. Compared with the NaCl treatment group, the contents of chlorophyll a (Chla) and chlorophyll b (Chlb) in walnut seedlings increased significantly by 63.44%, 93.02% and 72.26%, respectively, indicating that strain YBZ can significantly alleviate the damage of chlorophyll a, b and total chlorophyll in walnut leaves under salt stress (see Figure 4 ).

[0069] Example 7 Determination of proline content

[0070] As an osmotic regulating substance, proline can stabilize cell membranes, protect the integrity of cell membranes, participate in a variety of physiological and biochemical metabolic processes, and is a key substance for improving plant salt tolerance. The proline content of walnut leaves treated with different methods was determined by referring to the acid ninhydrin colorimetric method of Cao Jiankang et al.

[0071] Proline content (μg / g) = (C×V) / (W×a)

[0072] The results showed that the proline content of walnut seedlings in the NaCl treatment group was 25.54 μg / g, and the proline content of walnut seedlings in the strain YBZ4+NaCl treatment group reached 39.72 μg / g, which was significantly increased by 55.52% compared with the NaCl treatment group (see Figure 5 ), indicating that strain YBZ can significantly increase the content of proline in walnut seedlings under salt stress.

[0073] Example 8 Statistics of blade damage

[0074] The main symptom of walnut leaf scorch under salt stress is the appearance of scorch and wilting at the edge of the leaf, which then spreads to the center, eventually causing the entire leaf to become scorched and fall off in severe cases. Therefore, when developing a grading system for leaf scorch symptoms, the scorched leaf area of ​​walnut leaves is used as the basis for grading. It is divided into 0-4 levels according to the degree of damage, and the scorched leaf area of ​​each seedling leaf is counted to calculate the severity of damage to the seedling leaves. The grading standards for leaf scorch symptoms are shown in Table 3:

[0075] Table 3 Grading standards for leaf scorch symptoms in walnut seedlings

[0076]

[0077]

[0078] The damaged area of ​​leaves of each seedling was counted after NaCl stress, and the severity of leaf damage of walnut seedling scorch was calculated according to the following formula.

[0079] Damage severity = 100 × ∑ (number of damaged leaves at each level × representative value at each level) / (total number of leaves surveyed × highest representative value).

[0080] Control effect (%) = [1-(leaves injury severity of the strain and NaCl treatment group / leaves injury severity of the NaCl treatment group)] × 100%.

[0081] The results showed that the leaf damage severity of the NaCl treatment group was 79.80; the walnut leaf scorch symptoms of the strain YBZ4 and NaCl treatment groups were significantly alleviated compared with the NaCl treatment group, and the damage severity was only 5.17 (see Figure 6 , Figure 7 ), indicating that strain YBZ4 can significantly reduce the severity of damage caused by salt stress to walnut seedlings and effectively alleviate the occurrence of walnut leaf scorch under salt stress, with a control effect of 93.52% (see Figure 8 ).

[0082] In summary, the application of strain YBZ4 in the present invention can significantly reduce the damage caused by salt stress to walnut seedlings, improve the salt tolerance of walnut seedlings, and effectively alleviate the occurrence of scorch leaf disease of walnut seedlings under salt stress.

[0083] Although the present invention has been illustrated and described with specific embodiments, it should be appreciated that the above embodiments are only used to illustrate the technical solutions of the present invention rather than to limit it. Those skilled in the art should understand that the technical solutions described in the above embodiments may be modified, or some or all of the technical features thereof may be replaced by equivalents without departing from the spirit and scope of the present invention. However, these modifications or replacements do not make the essence of the corresponding technical solutions deviate from the scope of the technical solutions of the embodiments of the present invention. Therefore, this means that all such replacements and modifications within the scope of the present invention are included in the appended claims.

Claims

1. A strain of Pantoea rodasiiYBZ4 for preventing and treating walnut leaf scorch disease was deposited in the General Microbiology Center of China Microorganism Culture Collection Administration with the deposit number of CGMCC No.31371.

2. A microbial preparation, characterized in that: The microbial preparation includes PantoearodasiiYBZ4 described in claim 1.

3. The microbial preparation according to claim 2, characterized in that The microbial preparation comprises the bacteria of Pantoea rodasiiYBZ4 and / or its bacterial suspension.

4. The microbial preparation according to claim 2, characterized in that The microbial preparation includes: a solid preparation or a liquid preparation.

5. The microbial preparation according to claim 4, characterized in that When the microbial preparation is a solid preparation, the number of viable bacteria of Pantoea rodasii YBZ4 contained in the microbial preparation is 1.19×10 8 ~1.58×10 8 CFU / g; And / or, when the microbial preparation is a liquid preparation, the number of viable bacteria of Pantoea rodasii YBZ4 contained in the microbial preparation is 1.19×10 8 ~1.58×10 8 CFU / mL.

6. A method for preventing and treating walnut leaf scorch, characterized in that: The Pantoearodasii YBZ4 according to claim 1 and / or the microbial preparation according to any one of claims 2 to 5 are applied to walnuts.

7. The method for preventing and treating peach scorch according to claim 6, wherein: The application method includes: root irrigation.

8. Use of Pantoea rodasiiYBZ4 as claimed in claim 1 or the microbial preparation as claimed in any one of claims 2 to 5 in preventing and controlling walnut diseases.

9. The use according to claim 8, characterized in that: The walnut diseases include: diseases caused by salt stress.

10. The use according to claim 8, characterized in that: The walnut diseases include: walnut leaf scorch.

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