A strain of Pseudomonas paleroni and its application in plant disease control and promotion of phosphorus and iron absorption

Abstract

The present invention discloses a strain of Pseudomonas palleroniana and its application in plant disease prevention and control and promoting phosphorus and iron absorption. The strain is Pseudomonas palleroniana F1038, which has been deposited in Guangdong Provincial Microbial Culture Collection Center (GDMCC) on May 24, 2023, with a deposit number of GDMCC No: 63498. The Pseudomonas palleroniana F1038 of the present invention can significantly inhibit the growth of four plant disease pathogens, while being able to dissolve organic phosphorus and inorganic phosphorus and produce siderophores. Therefore, the Pseudomonas palleroniana F1038 of the present invention has great application potential in preventing and controlling plant pathogens and promoting plant phosphorus and iron absorption.

Description

Technical Field

[0001] The present invention relates to the technical field of microorganisms, in particular to a strain of Pseudomonas paleroni and application thereof in preventing and controlling plant diseases and promoting phosphorus and iron absorption. Background Art

[0002] Plant diseases are generally classified into bacterial diseases, fungal diseases, and viral diseases based on the differences in pathogens and symptoms. Among bacterial diseases, citrus canker caused by Xanthomonas citrisubsp.citri (Xcc) can cause citrus leaves to rot, branches to dry up, and even fruit deformity and rot, resulting in reduced citrus yield and quality. Ralstonia solanacearum is the second most serious plant pathogen in the world. It can cause devastating bacterial wilt of many important crops, among which tomato bacterial wilt is currently the focus of research and prevention. Rhodococcus fascians, as the only plant pathogen in the genus Rhodococcus, can cause tissue proliferation on the surface of plant leaves, forming leaf galls, which seriously affect plant growth and development. Pseudomonas syringae is a widely distributed Gram-negative rod-shaped bacterium that can infect a variety of crops, including beans, tomatoes, cauliflower, watermelon, alfalfa, and tobacco, causing lesions on the leaves and other parts of the plants, endangering their healthy growth. To cope with the increasingly serious bacterial diseases, synthetic chemical pesticides and antibiotics are widely used in plant disease prevention and control. However, the long-term and large-scale use of these chemicals has caused a series of problems such as environmental pollution and pesticide residues, and also threatens the quality and safety of agricultural products. More importantly, the long-term use of antibiotics can easily lead to the development of drug resistance and tolerance in crop pathogens, seriously reducing the effectiveness of drug application and indirectly threatening human life, health and safety. Therefore, the development of new green control methods for bacterial diseases is of great significance.

[0003] Obtaining highly effective antagonistic strains is fundamental to developing new, green methods for the prevention and control of bacterial diseases. Pseudomonas is widely distributed in plants and their surroundings, and it reproduces rapidly, has strong colonization ability, simple nutritional requirements, strong resistance to plant diseases, and significant effects in promoting plant growth. Therefore, it is often used for the biological control of bacterial diseases of plants. Currently, widely reported Pseudomonas species with biocontrol effects include Pseudomonas aeruginosa, Pseudomonas fluorescens, and Pseudomonas chlororaphis. As for Pseudomonas palleroniana, reported studies have focused on its control of melon seedling damping-off disease, tobacco black shank disease, pepper blight, and tea anthracnose. However, there are currently no reports on the use of Pseudomonas palleroniana in controlling plant diseases such as citrus canker, bacterial wilt, leaf gall, and leaf spot, or in promoting plant phosphorus and iron absorption. Summary of the Invention

[0004] The first object of the present invention is to overcome the shortcomings and deficiencies of the prior art and provide a strain of Pseudomonas, named Pseudomonas palleroniana F1038. The strain was deposited on May 24, 2023, at the Guangdong Provincial Microbiological Culture Collection Center (GDMCC), located at 5th Floor, Building 59, No. 100 Xianlie Middle Road, Yuexiu District, Guangzhou City, Guangdong Province, Postal Code: 510070, with a deposit number of GDMCC No: 63498.

[0005] The morphological characteristics of the Pseudomonas paleroni F1038 are as follows: Gram staining is negative, single colonies are round, light yellow, with a smooth, opaque surface and regular edges.

[0006] The second object of the present invention is to provide the use of the Pseudomonas paleroni F1038 in preventing and controlling plant diseases.

[0007] Preferably, the plant diseases include citrus canker, tomato bacterial wilt, plant leaf gall and leaf spot.

[0008] The third object of the present invention is to provide the use of the Pseudomonas paleronisi F1038 in promoting plant phosphorus and iron absorption.

[0009] The fourth object of the present invention is to provide the use of the Pseudomonas paleronisi F1038 in the preparation of biological preparations for preventing and controlling plant diseases and / or promoting plant phosphorus and iron absorption.

[0010] Preferably, the biological agent is a microbial agent, a microbial fertilizer, a microbial pesticide or a microbial additive.

[0011] Preferably, the plant diseases include citrus canker, tomato bacterial wilt, plant leaf gall and leaf spot.

[0012] A fifth object of the present invention is to provide a biological preparation for preventing and controlling plant diseases and / or promoting plant phosphorus and iron absorption, comprising Pseudomonas paleronisi F1038 or its fermentation liquid as an active ingredient.

[0013] A sixth object of the present invention is to provide a method for preventing and controlling citrus canker, tomato bacterial wilt, plant leaf gall and / or leaf spot, which comprises spraying the fermentation liquid of Pseudomonas paleroni F1038 to the area to be prevented and controlled.

[0014] The seventh object of the present invention is to provide a method for promoting plant phosphorus and iron absorption, which comprises applying Pseudomonas paleronis F1038 to plant cultivation to promote plant phosphorus and iron absorption.

[0015] The present invention has the following advantages and effects compared to the prior art:

[0016] This study reports for the first time that Pseudomonas paleronis F1038 has excellent efficacy against citrus canker, tomato bacterial wilt, plant leaf gall, and leaf spot. This strain can solubilize both organic and inorganic phosphorus, promoting phosphorus absorption in plants, and also produces siderophores, facilitating iron absorption in plants. The discovery and application of this strain's novel functions enrich my country's resources for highly effective disease prevention and growth promotion microorganisms, and holds great promise for applications in plant disease control and growth promotion.

[0017] Pseudomonas palleroniana F1038 was deposited on May 24, 2023 in the Guangdong Provincial Microbiological Culture Collection Center (GDMCC), located at 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou City, Guangdong Province, Postal Code: 510070, with the deposit number: GDMCC No: 63498. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1 This is the colony morphology of Pseudomonas paleroni F1038 on NA medium.

[0019] Figure 2 Phylogenetic tree of Pseudomonas paleroni F1038 based on the 16S rRNA gene.

[0020] Figure 3 The inhibitory effect of Pseudomonas paleroni F1038 on Xanthomonas citri subsp. citri (A), Ralstonia solanacearum (B), Rhodococcus spp. (C) and Pseudomonas syringae (D).

[0021] Figure 4Tests on the solubilization of organic and inorganic phosphorus and production of siderophores by Pseudomonas paleronisi F1038. DETAILED DESCRIPTION

[0022] The present invention will be further described below with reference to the accompanying drawings and specific examples, but the examples do not limit the present invention in any way. Unless otherwise specified, the reagents, methods and equipment used in the present invention are conventional reagents, methods and equipment in the art.

[0023] Example 1: Isolation and identification of Pseudomonas paleroni F1038

[0024] Healthy fruit trees in the citrus orchard of Fuzhuying Group, Xiaying Village, Huangkeng Town, Renhua County, Shaoguan City, Guangdong Province (N 25°4′17″, E 113°46′50″) were selected, and fresh citrus branches and leaves were collected using sterile scissors and placed in sterile sampling bags. Subsequently, the leaf samples were stored in a 4°C sampling box. Within 24 hours, the samples were brought back to the laboratory, the leaves were cut on the clean bench, and 10 g of leaves were weighed in a 500 mL triangular flask filled with 190 mL of sterile PBS buffer (0.2 mol / L, pH = 7.0). After shaking, it was placed in a 28°C constant temperature shaker and shaken at 200 rpm for 30 minutes. Subsequently, the triangular flask was placed in an ultrasonic oscillator at 40KHz, working for 8 seconds, resting for 2 seconds, and ultrasonicating for 10 minutes. Take 1 mL of the eluate and add it to a test tube containing 9 mL of PBS buffer, and dilute it to 10 -1 , 10 -2 , 10 -3 and 10 -4 , draw the original solution of eluent, 10 -1 , 10 -2 , 10 -3 and 10 -4 100 μL of each diluted sample was spread onto R2A medium (Qingdao Haibo, Catalog No. HB0167) and incubated in a 28°C biochemical incubator for 7 days. Based on characteristics such as colony size, color, dryness, smoothness, and the presence of halos, individual colonies were selected from the culture medium and transferred to fresh TSA medium (Qingdao Haibo, Catalog No. HB0177). Purification was performed by multiple streaking until a pure culture was obtained. The purified strains were numbered, mixed with 25% glycerol (v / v), and stored in an ultra-low temperature freezer, resulting in strain F1038.

[0025] The strain F1038 was Gram-negative. The strain was cultured in NA medium (Qingdao Haibo, product number HB0109) for 4 days. The colony morphology was as follows: Figure 1 As shown, the single colony is round, 2 to 4 mm in diameter, light yellow, with a smooth, opaque surface and neat edges.

[0026] Example 2: 16S rRNA gene sequence analysis of Pseudomonas paleroni F1038

[0027] The genomic DNA of strain F1038 was extracted and purified using the HiPure bacterial DNA extraction kit (Cat. No. D3146-03, Guangzhou Meiji Biotechnology Co., Ltd.). Using the purified DNA as a template, the bacterial 16S rRNA gene amplification universal primers 27F / 1492R, i.e. 27F (5'-AGAGTTTGATCCTGGCTCAG-3') and 1492R (5'-GGTTACCTTGTTACGACTT-3') were used to amplify the 16S rRNA gene sequence of strain F1038. After the amplification was completed, an appropriate amount of PCR product was taken for gel electrophoresis detection. When the presence of the target band was confirmed, the remaining PCR product was sent to Suzhou Jinweizhi Biotechnology Co., Ltd. for sequencing. The sequence is shown in SEQ ID NO.1, with a length of 1401bp. The 16SrRNA gene sequence of strain F1038 was submitted to the EzBioCloud database (www.ezbiocloud.net) for sequence homology comparison. The comparison results showed that strain F1038 and the model strain Pseudomonas palleroniana CFBP 4389 T The 16S rRNA gene sequence of the strain (accession number, AY091527) has the highest similarity of 99.8%; T and Pseudomonas lurida LMG 21995 T The similarity with the 16S rRNA gene sequence of the genus Pseudomonas (accession number, AF374472 and PDJB01000001) is second, both 99.6%; the similarity with the 16S rRNA gene of other type strains of the genus Pseudomonas is no higher than 99.5%. Figure 2 As shown, 16S rRNA gene similarity analysis indicated that strain F1038 shared the closest phylogenetic relationship with Pseudomonas palleroniana, and was therefore identified as Pseudomonas palleroniana. Strain F1038 was designated Pseudomonas palleroniana F1038. This strain was deposited on May 24, 2023, at the Guangdong Provincial Microbiological Culture Collection Center (GDMCC), Building 59, 5th Floor, No. 100 Xianlie Middle Road, Guangzhou, Guangdong Province, China, with the deposit number GDMCC No. 63498.

[0028]

[0029] Example 3: Identification of the inhibitory effect of Pseudomonas paleroni F1038 on plant pathogens

[0030] The punch method was used to test the inhibitory effect of Pseudomonas paleronis F1038 on four plant pathogens. The four plant pathogens (citrus canker pathogen Xcc, Ralstonia solanacearum GMI 1000, Rhodococcus diagenesis GDMCC1.839, and Pseudomonas syringae GDMCC 1.330) and Pseudomonas paleronis F1038 were inoculated into NB liquid medium for activation, and the fermentation broth was collected after 24 hours of culture. The fermentation broth of the four plant pathogens was spread on NA medium, and holes were punched in these media using a hole puncher. Subsequently, 50 μL of Pseudomonas paleronis F1038 fermentation broth was added to the wells, cultured at 28°C for 48 hours, and the diameter of the inhibition zone was measured using a vernier caliper.

[0031] like Figure 3 As shown, four plant pathogens formed clear zones of inhibition around the wells containing fermentation broth of Pseudomonas paleronis F1038. The diameters of the zones against citrus canker pathogen Xcc, Ralstonia solanacearum GMI 1000, Rhodococcus divaricata GDMCC1.839, and Pseudomonas syringae GDMCC 1.330 were 13.00, 17.00, 20.00, and 14.67 mm, respectively. These inhibition experiments demonstrated that Pseudomonas paleronis F1038 significantly inhibited the growth of citrus canker pathogen Xcc, Ralstonia solanacearum, Rhodococcus divaricata, and Pseudomonas syringae. Therefore, it is promising for the control of citrus canker, tomato bacterial wilt, plant leaf gall, and leaf spot diseases.

[0032] Example 4: Identification of phosphate solubilization and siderophore production by Pseudomonas paleroni F1038

[0033] Activated Pseudomonas paleronis F1038 was inoculated into organophosphate medium (Qingdao Haibo, product number HB8673) and inorganic phosphorus medium (Qingdao Haibo, product number HB8670), cultured at 28°C for 7 days, and the diameter of the hydrolysis zone was measured using a vernier caliper. Activated Pseudomonas paleronis F1038 was inoculated into R2A medium and cultured at 28°C for 48 hours. Subsequently, CAS detection medium (Qingdao Haibo, product number HB9132) was poured over the R2A medium and cultured at 28°C for 48 hours. The diameter of the siderophore secretion zone (orange) was measured using a vernier caliper.

[0034] like Figure 4As shown, Pseudomonas paleronisi F1038 formed clearly visible hydrolysis zones with diameters of 33.7 and 15 mm on both organophosphate and inorganic phosphate media, respectively. In CAS assay medium, a clearly visible orange-yellow siderophore secretion zone with a diameter of 31.3 mm formed around Pseudomonas paleronisi F1038. These test results demonstrate that Pseudomonas paleronisi F1038 can decompose both organic and inorganic phosphate, increasing the available phosphorus content in the surrounding environment and promoting phosphorus absorption by plants. The siderophores it secretes can efficiently bind to iron in the surrounding environment, forming stable chelates with it, thereby effectively absorbing low-soluble iron in the environment and promoting plant growth.

[0035] These results demonstrate that Pseudomonas palleroniana F1038 is a highly effective biocontrol strain with significant inhibitory effects on the pathogens of citrus canker (Xcc), tomato bacterial wilt (Raulia solanacearum), plant leaf gall (Rhodococcus diagenesis), and plant leaf spot (Pseudomonas syringae). This strain can be used directly for the prevention and control of citrus canker, tomato bacterial wilt, plant leaf gall, and leaf spot through spraying, or in the production of microbial preparations and fertilizers, potentially for the prevention and control of citrus canker, tomato bacterial wilt, plant leaf gall, and leaf spot. Furthermore, Pseudomonas palleroniana F1038 can solubilize organic and inorganic phosphorus and produce siderophores, making it suitable for use in the production of microbial preparations and fertilizers to promote phosphorus and iron absorption in plants.

[0036] The above are merely preferred embodiments of the present invention. It should be noted that the above preferred embodiments should not be construed as limiting the present invention, and the scope of protection of the present invention should be determined by the scope defined in the claims. Persons skilled in the art will appreciate that improvements and modifications may be made without departing from the spirit and scope of the present invention, and such improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. A strain of Pseudomonas paleronis ( Pseudomonas palleroniana ) F1038, deposit number: GDMCC No: 63498.

2. Use of the Pseudomonas paleronis F1038 according to claim 1 in preventing and controlling plant diseases, wherein the plant diseases include citrus canker, tomato bacterial wilt, plant leaf gall and leaf spot; the pathogen of citrus canker is Xanthomonas citri subsp. citri Xanthomonas citri subsp. citri ; The pathogen of tomato bacterial wilt is Ralstonia solanacearum; the pathogen of plant leaf gall disease is Rhodococcus spp.; the pathogen of leaf spot disease is Pseudomonas syringae.

3. Use of the Pseudomonas paleronisi F1038 according to claim 1 in promoting phosphorus and iron absorption in plants.

4. Use of the Pseudomonas paleronis F1038 according to claim 1 in the preparation of a biological agent for preventing and treating plant diseases and / or promoting plant phosphorus and iron absorption, wherein the plant diseases include citrus canker, tomato bacterial wilt, plant leaf gall and leaf spot; the pathogen of citrus canker is Xanthomonas citri subsp. citri Xanthomonas citri subsp. citri ; The pathogen of tomato bacterial wilt is Ralstonia solanacearum; the pathogen of plant leaf gall disease is Rhodococcus spp.; the pathogen of leaf spot disease is Pseudomonas syringae.

5. The use according to claim 4, characterized in that The biological agent is a microbial agent, a microbial fertilizer, a microbial pesticide or a microbial additive.

6. A biological agent for preventing and controlling plant diseases and / or promoting plant phosphorus and iron absorption, characterized in that: The invention contains the Pseudomonas paleronis F1038 or its fermentation liquid as claimed in claim 1 as an active ingredient; the plant diseases include citrus canker, tomato bacterial wilt, plant leaf gall and leaf spot; the pathogen of citrus canker is Xanthomonas citri subsp. Xanthomonas citri subsp. citri ; The pathogen of tomato bacterial wilt is Ralstonia solanacearum; the pathogen of plant leaf gall disease is Rhodococcus spp.; the pathogen of leaf spot disease is Pseudomonas syringae.

7. A method for preventing and controlling citrus canker, tomato bacterial wilt, plant leaf gall and / or leaf spot, characterized in that: The fermentation liquid of Pseudomonas paleroni F1038 of claim 1 is sprayed to the area to be controlled; the pathogen of citrus canker is Xanthomonas citri subsp. Xanthomonas citri subsp. citri ; The pathogen of tomato bacterial wilt is Ralstonia solanacearum; the pathogen of plant leaf gall disease is Rhodococcus spp.; the pathogen of leaf spot disease is Pseudomonas syringae.

8. A method for promoting plant phosphorus and iron absorption, characterized in that: The method comprises applying the Pseudomonas paleroni F1038 according to claim 1 to plant cultivation.

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