Pantoea rodasii YBZ4 for preventing and treating walnut leaf scorch and application thereof

Abstract

The present application relates to the field of microbial technology, and in particular, to a Pantoea rodasii YBZ4 for preventing and treating walnut leaf scorching and application thereof. The Pantoea rodasii YBZ4 for preventing and treating walnut leaf scorching is preserved in the China General Microbiological Culture Collection Center, and the preservation number is CGMCC No.31371. The Pantoea rodasii YBZ4 can effectively reduce the occurrence of walnut seedling leaf scorching, the damage degree of walnut seedling leaves under salt stress is significantly reduced, and the prevention and treatment effect can reach more than 90%.

Description

Technical Field

[0001] This invention relates to the field of microbial technology, and more specifically, to a Pantoearodasii YBZ4 strain for the prevention and treatment of walnut leaf scorch and its applications. Background Technology

[0002] Walnut (Juglans regia L.) is a plant belonging to the genus Juglans in the family Juglandaceae, distributed in Northwest, Southwest, North, and South my country. Juglans leaf necrosis (JLN) is a physiological disorder that begins with scorching at the leaf margins, then extends towards the center, gradually spreading to the entire leaf. Affected walnut plants exhibit browning and scorching of leaf margins, inhibited photosynthesis, and blackened, underdeveloped fruit, severely impacting walnut yield and quality. This disease was first reported in the main walnut-producing area of ​​southern Xinjiang in 2012, and in recent years, the affected area has shown a trend of increasing annually, earlier onset, and greater severity. Juglans leaf necrosis may be caused by high temperatures and hot, dry winds leading to intense transpiration, with transpiration exceeding absorption, rapid evaporation of surface water, low rainfall, and insufficient irrigation, resulting in leaf scorching. It may also be due to the presence of high levels of sodium (Na) in saline soils. + and Cl - Walnut leaf scorch can lead to the accumulation of toxic ions in walnut leaves, disrupting ion homeostasis and causing ion toxicity, which damages leaf function and causes leaf scorch. Furthermore, weak tree vigor, soil compaction, or inadequate salt and alkali removal measures can also easily trigger walnut leaf scorch. Walnut leaf scorch severely impacts fruit yield and quality, causing yield reductions of over 60% in some orchards. In severe cases, it can even lead to tree weakness and death, seriously threatening the healthy development of the Xinjiang walnut industry. Therefore, developing practical and feasible prevention and control measures and carrying out effective control of walnut leaf scorch is urgently needed.

[0003] Traditional methods of soil improvement mainly include constructing drainage ditches, surface irrigation to leach soil salinity, lowering the water table, improving soil structure, strengthening plant pruning, and improving light conditions. However, these measures are labor-intensive, resource-intensive, and slow to take effect, failing to effectively solve the problem. In recent years, spraying anti-seepage agents to reduce water loss from walnut trees and applying organic fertilizers such as humic acid and potassium fulvate to increase soil organic matter content have effectively controlled walnut leaf scorch, but their economic costs are high. Beneficial rhizosphere bacteria have functions such as phosphorus solubilization, potassium solubilization, and nitrogen fixation. They can also secrete IAA and ACC deaminases and produce various substances such as siderophores. By regulating plant ion homeostasis and reducing oxidative damage, they alleviate abiotic stress on plants, making them an important approach to achieving green control of walnut leaf scorch. The discovery of beneficial rhizosphere bacteria that alleviate walnut leaf scorch and the development and application of beneficial microbial preparations for alleviating walnut leaf scorch have attracted widespread attention.

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

[0005] The purpose of this invention is to provide a Pantoea rodasii YBZ4 plant for preventing and treating walnut leaf scorch and its application. The Pantoea rodasii YBZ4, isolated from the rhizosphere soil of walnut, can alleviate the damage caused by salt stress to walnuts and has the effect of preventing and treating walnut leaf scorch.

[0006] In order to achieve the above-mentioned objectives of the present invention, the following technical solution is adopted:

[0007] One aspect of the present invention relates to a Pantoea rodasii YBZ4 strain for the prevention and treatment of walnut leaf scorch, which is deposited at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC No. 31371.

[0008] The Pantoea rodasii YBZ4, isolated from walnut rhizosphere soil, can improve the growth ability of walnuts under salt stress, has the characteristic of rapid colonization in walnut roots, and has a good control effect on walnut leaf scorch, showing good application prospects.

[0009] Another aspect of the invention relates to a microbial preparation comprising Pantoea rodasii YBZ4 as described in claim 1.

[0010] The aforementioned microbial preparation contains Pantoea rodasii YBZ4 as its active ingredient. It is a beneficial plant microbial preparation that can be used to reduce the occurrence of walnut leaf scorch. This microbial preparation has a good control effect on walnut leaf scorch, a short onset time after application, reduces the control cost of walnut leaf scorch, and is environmentally friendly. This microbial preparation can also be used in combination with physical and chemical control methods to further improve the control effect of walnut leaf scorch.

[0011] Furthermore, the microbial preparation comprises the cells of Pantoea rodasii YBZ4 and / or its bacterial suspension. Verification by this invention has shown that both the cells of Pantoea rodasii YBZ4 and its bacterial suspension have good preventative effects against walnut leaf scorch.

[0012] This invention does not specifically limit the dosage form of the microbial preparation; conventional dosage forms in the art can be applied to the technical solutions of this invention. In some specific embodiments, the microbial preparation includes, but is not limited to, solid or liquid formulations.

[0013] Furthermore, when the microbial preparation is a solid dosage form, the number of viable Pantoearodasii YBZ4 bacteria in the microbial preparation is 1.19 × 10⁻⁶. 8 ~1.58×10 8 CFU / g.

[0014] Furthermore, when the microbial preparation is a liquid preparation, the number of viable Pantoearodasii YBZ4 bacteria in the microbial preparation is 1.19 × 10⁻⁶. 8 ~1.58×10 8 CFU / mL.

[0015] Limiting the number of viable Pantoea rodasii YBZ4 bacteria in microbial agents to a certain range can ensure that the microbial agents exert the best control effect on walnut leaf scorch and provide theoretical dosage guidance for the practical application of Pantoea rodasii YBZ4.

[0016] Furthermore, the microbial preparation also includes excipients conventional in the art.

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

[0018] This invention does not specifically limit the application method; conventional application methods in the art can be used to implement the technical solution of this invention. In some specific embodiments, the application method includes, but is not limited to, root irrigation.

[0019] Another aspect of the present invention relates to the use of the Pantoea rodasii YBZ4 or the microbial preparations described herein in the prevention and control of walnut diseases.

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

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

[0022] Preservation information for strain YBZ4: Pantoena rodasii YBZ4 was deposited on July 19, 2024, at the China General Microbiological Culture Collection Center (CGMCC), with accession number CGMCC No. 31371. Address: Institute of Microbiology, Chinese Academy of Sciences, No. 3, No. 1 Beichen West Road, Chaoyang District, Beijing, 100101, China.

[0023] Compared with the prior art, the beneficial effects of the present invention are as follows:

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

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

[0026] To more clearly illustrate the specific embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the specific embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are some embodiments of the present invention. For those skilled in the art, other drawings can be obtained from these drawings without creative effort.

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

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

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

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

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

[0032] Figure 6 To illustrate the effect of strain YBZ4 on alleviating leaf scorch 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: 5cm).

[0033] Figure 7 To alleviate the severity of leaf damage in walnut seedlings caused by strain YBZ4;

[0034] Figure 8 The study investigated the control effect of strain YBZ4 on walnut leaf scorch. Detailed Implementation

[0035] The technical solution of the present invention will be clearly and completely described below with reference to the accompanying drawings and specific embodiments. However, those skilled in the art will understand that the embodiments described below are some embodiments of the present invention, but not all embodiments, and are only used to illustrate the present invention, and should not be regarded as limiting the scope of the present invention. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention. Where specific conditions are not specified in the embodiments, conventional conditions or conditions recommended by the manufacturer shall be followed. Where the manufacturers of reagents or instruments are not specified, they are all conventional products that can be purchased commercially.

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

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

[0038] Genomic DNA was extracted from strain YBZ4 and used as a template. PCR amplification was performed using universal primers 27F: 5'-AGAGTTTGATCMTGGCTCAG-3' and 1492R: 5'-TACGGYTACCTTGTTACG ACTT-3', according to the reaction system shown in 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 subjected to a BLAST search at the National Center for Biotechnology Information (NCBI) in the United States. The sequence with the highest similarity to the 16S rRNA gene fragment 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 at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC No. 31371 on July 19, 2024.

[0047] (2) Colony morphology of strain YBZ4

[0048] After inoculating strain YBZ4 onto LB solid medium and incubating at 30°C for 24 hours, the colonies were observed to be pale yellow, with a raised center, and smooth and moist surfaces and edges (see...). Figure 1 ).

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

[0050] The physiological and biochemical characteristics of strain YBZ4 were mainly determined according to the "Handbook of Systematic Identification of Common Bacteria". Culture media were prepared to test the ability of strain YBZ4 to secrete methyl red, amylase, protease, lipase, catalase, hydrogen peroxide, and produce IAA. No hydrolysis zone was observed in protein solid medium and fat medium, indicating that the strain cannot secrete protease and lipase and cannot hydrolyze proteins and fats. A hydrolysis zone was observed in starch medium, indicating that the strain has the ability to hydrolyze starch. Strain YBZ4 was negative for methyl red, but positive for hydrogen peroxide, catalase, and IAA. The results of the physiological and biochemical characteristics of strain YBZ4 are shown in Table 2.

[0051] Table 2. Detection of physiological and biochemical characteristics of strain YBZ4

[0052]

[0053] +: The presence of a hydrolysis zone or color reaction indicates that the bacteria can secrete the substance; -: The absence of a hydrolysis zone or color reaction indicates that the bacteria cannot secrete the substance.

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

[0055] The YBZ4 strain preserved in Example 2 was activated on solid LB medium and cultured until single colonies were produced. A single colony was picked and transferred to 5 mL of liquid LB medium and incubated overnight at 30°C and 200 rpm. The next day, the entire bacterial culture was transferred to an Erlenmeyer flask containing 200 mL of liquid LB medium and incubated overnight at 30°C and 200 rpm. Then, the entire bacterial culture was centrifuged at 6000 rpm for 10 min, the supernatant was discarded, and the bacterial precipitate was retained. The bacterial precipitate was resuspended in sterile water, and the bacterial concentration was adjusted to obtain a final concentration of 1.19 × 10⁻⁶ in 200 mL. 9 ~1.58×10 9 The bacterial suspension at CFU / mL was diluted with 800 mL of sterile water to a final concentration of 1.19 × 10⁻⁶. 8 ~1.58×10 8 1L of bacterial suspension with CFU / mL.

[0056] Example 3: Walnut seedling salt tolerance promotion experiment in greenhouse

[0057] After the walnut seedlings germinate, water them with clean water every 4 days, using 80mL of water per pot, maintaining suitable humidity. Approximately 15 days after germination, conduct an inoculation test with salt-tolerant bacteria. The seedling standards for the test are as follows:

[0058] Seedlings with a height >15cm and a total number of leaves >10.

[0059] The bacterial inoculation method was as follows: A bacterial suspension of strain YBZ4 was obtained using the method described in Example 2. Seedlings that had germinated and grown to a height >15cm and had more than 10 leaves were selected. Strain YBZ4 was inoculated every 4 days via root drenching, with 80mL of bacterial suspension per pot, for a total of 3 inoculations. The NaCl treatment group and the water control group were simultaneously irrigated with an equal amount of water. After inoculation, salt treatment could begin. The method was as follows: the NaCl control group and the strain YBZ4 + NaCl treatment group were irrigated with 80mL of 75mM NaCl solution every 4 days, while the water control group was simultaneously irrigated with an equal amount of water, for a total of 6 irrigations.

[0060] Example 4: Determination of malondialdehyde content

[0061] Malondialdehyde (MDA), a product of cell membrane lipid peroxidation, exacerbates cell membrane damage. This experiment used a MDA content kit (Suzhou Grease Biotechnology Co., Ltd.) to detect the MDA content in the leaves of walnut seedlings under different treatments.

[0062] The results showed that the malondialdehyde (MDA) content in walnut seedlings treated with NaCl was 24.93 nmol / g, while the MDA content in the YBZ4 + NaCl treatment group was only 12.65 nmol / g, a decrease of 49.26% compared to the NaCl treatment group. Although slightly higher than the water control group, the difference was not statistically significant, indicating that inoculation with strain YBZ4 could significantly reduce the degree of membrane damage in the leaves of salt-stressed walnut seedlings (see...). Figure 2 ).

[0063] Example 5: Determination of peroxidase content

[0064] Catalase activity can evaluate the degree of reactive oxygen species (ROS) toxicity in plants; the higher the catalase content, the less toxic the plant is under stress. This experiment used a catalase content kit (Suzhou Grease Biotechnology Co., Ltd.) to detect the catalase content in walnut leaves under different treatments.

[0065] The results showed that the peroxidase content of walnut seedlings in the NaCl-treated group was 44.35 μmol / min / g. After inoculation with strain YBZ4 under salt stress, the peroxidase content in the leaves of walnut seedlings reached 94.55 μmol / min / g, which was significantly higher than that in the NaCl-treated group by 113.19%. This indicates that inoculation with strain YBZ4 can effectively alleviate the toxicity of reactive oxygen species to walnut seedling leaves (see...). Figure 3 ).

[0066] Example 6: Determination of chlorophyll content

[0067] Walnut leaves from different treatments were taken, quickly rinsed with sterile water, and then dried with filter paper. 0.3 g of each sample was weighed, and each treatment was repeated three times. The 0.3 g sample was placed in a pre-cooled mortar, and an appropriate amount of 95% ethanol solution was added to quickly homogenize the mixture. The homogenate was transferred to a 50 mL centrifuge tube, and 5 mL of 95% ethanol solution was added to wash the precipitate, repeating twice. The volume was adjusted to 25 mL, and the mixture was centrifuged at 9000 rpm for 10 min at 4 °C. After filtration, the supernatant was used to measure the absorbance at 645 nm and 663 nm.

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

[0069] Example 7: Determination of Proline Content

[0070] Proline, as an osmotic regulator, stabilizes cell membranes, protects their integrity, and participates in various physiological and biochemical metabolic processes, making it a key substance for improving plant salt tolerance. The proline content in walnut leaves under different treatments was determined using the acidic ninhydrin colorimetric method described by 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-treated group was 25.54 μg / g, while the proline content of walnut seedlings in the YBZ4+NaCl-treated group reached 39.72 μg / g, which was significantly increased by 55.52% compared with the NaCl-treated group (see...). Figure 5 This indicates that strain YBZ can significantly increase the proline content in walnut seedlings under salt stress.

[0073] Example 8: Statistical analysis of leaf damage

[0074] The main symptoms of walnut leaf scorch under salt stress are scorching and wilting at the leaf edges, which then spreads towards the center, eventually causing the entire leaf to become scorched and, in severe cases, to fall off. Therefore, when grading leaf scorch symptoms, the scorched leaf area of ​​the walnut leaf is used as the grading basis. The severity of damage is divided into grades 0-4, and the scorched leaf area of ​​each seedling is counted to calculate the severity of leaf damage. The grading standards for leaf scorch symptoms are shown in Table 3.

[0075] Table 3 Grading Standards for Leaf Scorch in Walnut Seedlings

[0076]

[0077]

[0078] After NaCl stress, the damaged area of ​​each seedling leaf was counted, and the severity of leaf damage in walnut seedlings with leaf scorch was calculated using the following formula.

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

[0080] Control efficacy (%) = [1 - (severity of leaf damage in strain and NaCl treatment group / severity of leaf damage in NaCl treatment group)] × 100%.

[0081] The results showed that the leaf damage severity in the NaCl-treated group was 79.80; the symptoms of walnut leaf scorch in the YBZ4 strain and NaCl-treated groups were significantly reduced compared to the NaCl-treated group, with a damage severity of only 5.17 (see...). Figure 6 , Figure 7 This indicates 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 this 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 leaf scorch in walnut seedlings under salt stress.

[0083] Although the present invention has been illustrated and described with specific embodiments, it should be understood that the above embodiments are only used to illustrate the technical solutions of the present invention, and not to limit them; those skilled in the art should understand that modifications can be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some or all of the technical features therein, without departing from the spirit and scope of the present invention; and these modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of the present invention; therefore, this means that all such substitutions and modifications that fall within the scope of the present invention are included in the appended claims.

Claims

1. A plant for preventing walnut leaf scorch Pantoea rodasii YBZ4 is deposited at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC No. 31371.

2. A microbial preparation, characterized in that, The microbial preparation includes the one described in claim 1. Pantoea rodasii YBZ4.

3. The microbial preparation according to claim 2, characterized in that, The microbial preparation includes the above. Pantoea rodasii YBZ4 cells and / or its bacterial suspension.

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

5. The microbial preparation according to claim 4, characterized in that, When the microbial preparation is a solid dosage form, the microbial preparation contains the... Pantoea rodasii The viable count of YBZ4 was 1.19 × 10⁻⁶. 8 ~1.58×10 8 CFU / g; And / or, when the microbial preparation is a liquid preparation, the microbial preparation contains the... Pantoea rodasii The viable count of YBZ4 was 1.19 × 10⁻⁶. 8 ~1.58×10 8 CFU / mL.

6. A method for preventing and controlling walnut leaf scorch, characterized in that, Apply the method of claim 1 to walnuts Pantoea rodasii YBZ4 and / or the microbial preparation according to any one of claims 2 to 5.

7. The method for preventing and controlling peach leaf scorch according to claim 6, characterized in that, The method of application is: root irrigation.

8. As described in claim 1 Pantoea rodasii The application of YBZ4 or any one of claims 2 to 5 in the prevention and control of walnut diseases; The walnut disease mentioned is caused by salt stress.

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