Pseudomonas in northwest province and application of pseudomonas in prevention and treatment of bacterial blight of rice

Abstract

The invention relates to pseudomonas in northwest province and application thereof in prevention and treatment of bacterial blight of rice, the strain is preserved in China General Microbiological Culture Collection Center, the address is No.3, No.1 yard, Beichen West Road, Chaoyang District, Beijing, the preservation number is CGMCC NO.25493, the preservation date is August 5, 2022, and the preservation number is CGMCC NO.25493. The strain can be used for preventing and treating bacterial leaf blight caused by xanthomonas oryzae pv. Oryzae pv. Oryzae, the effect is remarkable, the prevention effect in a pot experiment reaches 72%, meanwhile, the disease time can be delayed, and therefore the strain has the huge potential of being applied as a biopesticide.

Description

Technical Field

[0001] The present invention belongs to the field of crop disease control, and particularly relates to a Pseudomonas wayambapalatensis from the northwest province and its application in the control of rice bacterial blight. Background Art

[0002] Rice bacterial blight is one of the three major diseases of rice in China. Infected rice generally reduces production by 20% - 30%, and can reach 50% in severe cases. If the blight - withering type appears during the tillering stage, causing a large number of rice plants to wither and die, the loss will be even greater.

[0003] Although good results have been achieved in controlling rice bacterial blight with chemical agents, there are very few products for microbial control of rice bacterial blight, and the effects are not ideal enough. To achieve the biological control of rice bacterial blight, the present invention has carried out strain screening and corresponding control effect research. Summary of the Invention

[0004] To solve the above - mentioned technical problems, the present invention provides a strain which is preserved in the China General Microbiological Culture Collection Center, with the address being No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing. The preservation number is CGMCC NO.25493, and the preservation date is August 5, 2022. The strain belongs to the species Pseudomonas wayambapalatensis, and the strain is named FY - 19.

[0005] The present invention also provides the use of the above - mentioned strain, and the strain is used for controlling rice bacterial blight or inhibiting the growth of Xanthomonas oryzae pv. Oryzae.

[0006] Preferably, in the above - mentioned use, the rice bacterial blight is caused by Xanthomonas oryzae pv. Oryzae (Xoo), and the Xanthomonas oryzae pv. Oryzae is the causal agent of rice bacterial blight.

[0007] The present invention also provides an agricultural biological control bactericide, which is characterized in that the bactericide contains the cells of the above - mentioned strain.

[0008] Advantages of the Invention

[0009] The strain FY - 19 of the present invention has an obvious inhibitory effect on the growth of Xanthomonas oryzae pv. Oryzae and has a high antagonistic effect.

[0010] The strain FY-19 of the present invention can be applied to control bacterial blight caused by Xanthomonas oryzae pv. Oryzae (Xoo), with significant effects. The control efficacy reaches 72% in pot experiments, and at the same time, it can delay the onset time. Therefore, the strain of the present invention has great potential for application as a biological pesticide. Description of the Drawings

[0011] Figure 1 It is the antagonistic activity diagram of FY-19 bacterial liquid against Xanthomonas oryzae pv. Oryzae.

[0012] Figure 2 It is the colony characteristic diagram of strain FY-19.

[0013] Figure 3 It is the maximum likelihood tree (ML) constructed by 16S, gyrB, rpoB, and rpoD gene sequences, showing the taxonomic position of FY-19 in the Pseudomonas group of the northwest province. Detailed Implementation Modes

[0014] Preparation of suspension of Xanthomonas oryzae pv. Oryzae: Inoculate the Xoo pathogen into WA liquid medium and culture it at 30 °C for 24 h. Dilute the obtained bacterial liquid with WA medium to OD 600 = 0.25 (1×10 7 CFU / mL) to obtain the suspension of Xanthomonas oryzae pv. Oryzae, which is abbreviated as Xoo suspension in the following examples.

[0015] Example 1: Isolation and Screening of Strains

[0016] The test samples were divided into soil and rice leaf samples, all collected from the rice experimental fields with continuous cropping of the China National Rice Research Institute. The test samples were all collected from the periphery of the severely bacterial blight-infected areas. The samples were placed in sterile bags, sealed and stored in a 4 °C refrigerator.

[0017] Screening of biocontrol bacteria in samples by double-layer method

[0018] Soil samples: Take 1 g of soil sample and prepare it into a soil sample suspension in 9 mL of sterile water.

[0019] Double-layer method screening: Uniformly coat 1 mL of Xoo suspension on NA agar medium, dry and fix it in a laminar flow hood, then uniformly coat the second layer of 1 mL of soil sample suspension, dry and fix it in a laminar flow hood, and then incubate the agar plate in the dark at 28 °C for 3 d. Select the colonies with inhibition zones for purification culture, and then conduct re-screening.

[0020] A total of 5 strains with biocontrol and antibacterial effects were obtained through the above screening, and were named FY-18, FY-19, FY-20, FYW-13 and FYW-14 respectively.

[0021] Example 2: Antagonistic effect of strains against Xanthomonas oryzae pv. oryzae

[0022] The strains FY-18, FY-19, FY-20, FYW-13 and FYW-14 obtained from the primary screening were respectively inoculated into NB liquid medium (glucose 2.5 g / L, beef extract 3 g / L, peptone 10 g / L, sodium chloride 5 g / L, pH = 7.2), and cultured until OD 600 = 0.7, and the cell number was 2×10 9 CFU / mL, obtaining biocontrol bacterial solutions of strains FY-18, FY-19, FY-20, FYW-13 and FYW-14: FY-18 bacterial solution, FY-19 bacterial solution, FY-20 bacterial solution, FYW-13 bacterial solution and FYW-14 bacterial solution.

[0023] 500 μL of the Xoo suspension was evenly spread on the NA solid medium culture dish, dried and fixed in the ultra-clean workbench. A sterilized quantitative filter paper disc (Φ0.5 cm) was placed in the center of the culture dish and 10 μL of the above biocontrol bacterial solution was added dropwise. 10 μL of sterile water was added dropwise in another identical culture dish as a blank control. After the prepared plate was placed in the dark at 28°C for 24 h, the diameter of the inhibition zone was measured and recorded.

[0024] The inhibition effects of different biocontrol bacterial solutions are shown in Table 1 below. The results show that the antibacterial effect of strain FY-19 (see Appendix Figure 1 ) is the most significant.

[0025] Table 1 Antagonistic effect of biocontrol bacterial solution against Xanthomonas oryzae pv. oryzae

[0026] Biocontrol strain bacterial liquid Diameter of inhibition zone FY-18 bacterial liquid <![CDATA[2.28±0.21 bc > FY-19 bacterial liquid <![CDATA[3.01±0.08 a > FY-20 bacterial liquid <![CDATA[2.66±0.36 ab > FYW-13 bacterial liquid <![CDATA[2.72±0.45 a > FYW-14 bacterial liquid <![CDATA[2.08±0.85 c >

[0027] Example 3: Identification of strain FY-19

[0028] The strain FY-19 with the best screening effect was identified

[0029] Morphological characteristics

[0030] The colony surface of strain FY-19 on NA medium is smooth and milky white (see Appendix Figure 2 ). The staining test determined it to be a Gram-negative bacillus, which is one of the main groups of plant rhizosphere microorganisms. It has simple nutritional requirements, a fast proliferation rate and strong rhizosphere colonization ability.

[0031] 16S rDNA, gyrB, rpoB and rpoD sequence analysis

[0032] The FY-19 bacterial solution (the FY-19 bacterial solution prepared in Example 2) was subjected to DNA extraction. Using this as a template, the 16S rDNA gene of the strain was amplified with primers 27F and 1492R. After bidirectional sequencing of the amplification product, it was classified through the GenBank database. To distinguish the species boundaries between closely related strains within the same genus, phylogenetic analysis was performed using the gyrB, rpoB, and rpoD genes. The primers for the rpoD gene were 70F: 5’-ACGACTGACCCGGTACGCATGTAYATGMGNGARATGGGNACNGT-3’ and 70R: 5’-ATAGAAATAACCAGACGTAAGTTNGCYTCNACCATYTCYTTYTT-3’; the primers for the gyrB gene were UP-1: 5’-GAAGTCATCATGACCGTTCTGCAYGCNGGNGGNAARTTYGA-3’ and P-2r: 5’-AGCAGGGTACGGATGTGCGAGCCRTCNACRTCNGCRTCNGTCAT-3’; the primers for the rpoB gene were LAPS5: 5’-TGGCCGAGAACCAGTTCCGCGT-3’ and LAPS27: 5’-CGGCTTCGTCCAGCTTGTTCAG-3’. All PCR amplifications were carried out for 35 cycles, and each cycle included a denaturation step at 95 °C for 30 s, an annealing step between 50 - 55 °C for 30 s, and an extension step at 72 °C for 1 minute. After staining with the nucleic acid dye TS-GelRed (Qingke, China), on a 1% agarose gel, the amplification products were observed under an ultraviolet transilluminator, and the products were recovered, ligated to the T vector, transformed, positive clones were picked, plasmids were extracted, and bidirectional sequencing was performed. The sequences were edited using Clustalx1.83, and each sequence was compared with other sequences in the GenBank database using BLAST. Closely related genes were downloaded, and Pseudomonas aeruginosa was used as an outgroup. For phylogenetic analysis, the sequences of each gene were aligned using MAFFT 7.273, and the sequence matrix was trimmed using Gblocks 0.91b. A phylogenetic tree with maximum likelihood (ML) was constructed using RaxmlGUI v.1.5. The ML analysis used 1000 rapid bootstrap replicates, and a threshold of ≥50% was used as the cutoff value for significantly supported nodes.

[0033] BLAST analysis of the ribosomal 16S gene sequence showed that strain FY-19 had 99.93% identity with Pseudomonas taiwanensis BCRC 17751. The results of phylogenetic analysis showed that all isolated strains were divided into different branches (see attachment Figure 3 ), and the genetic relationships of most reference strains could be clearly distinguished at the species level. Among them, strain FY-19 clustered together with Pseudomonas wayambapalatensis to form a distinct branch with a bootstrap value of 100%. Therefore, through 16S gene sequence and phylogenetic analysis, strain FY-19 was identified as Pseudomonas wayambapalatensis.

[0034] Based on morphological characteristics, 16S rDNA, gyrB, rpoB, and rpoD gene sequences, and phylogenetic analysis, strain FY-19 was identified as Pseudomonas wayambapalatensis. This strain was deposited in the General Microbiological Center of the China Committee for Culture Collection of Microorganisms on August 5, 2022, at No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing. The deposit number was CGMCC NO. 25493, and the strain was named FY-19.

[0035] Example 4: Pot experiment on the control of rice bacterial blight by strain FY-19

[0036] A total of 2 experimental groups and 2 control groups were set up in the pot experiment. Each experimental group had 15 clumps as one treatment, and each treatment had 3 replicates. The rice variety was Zhongzao 39, and rice seedlings were raised in a greenhouse. When the rice plants grew to the tillering stage, the FY-19 bacterial liquid (the FY-19 bacterial liquid prepared in Example 2) was diluted 200 times. The time for spraying the bacterial agent on the leaves was after the rice was kept moist and exuded water. After keeping moist for 1 h, normal management was carried out. The control agent group sprayed a 1500-fold dilution of 20% thiazole zinc suspension, and the blank and control CK sprayed an equal amount of sterilized water. Three days after inoculating the fermentation broth, in an imitation of the natural infection method in the field, the rice seedlings were inoculated with Xoo without injury. The main method was to keep the above 4 treated rice seedlings moist and exude water. Except for the blank treatment, the remaining ones were sprayed with Xoo suspension, kept moist for 1 h, and the moisture-keeping film was removed to let the water-exuding phenomenon disappear. The treated rice grew under the same conditions. Four days, 8 days, 14 days, and 25 days after inoculating the bacteria, the symptoms were observed, and the disease severity and control effect were counted.

[0037] The method for counting the disease severity was evaluated according to the IRRI-2013 grading standard of the International Rice Research Institute, that is, based on the leaf veins, it was divided into 9 parts (8 leaf veins), but the two sides could be combined as 1 part; that is, the leaf was divided into 8 parts.

[0038] Grade 0: No lesions

[0039] Grade 1: < 1%; Lesions appear at 1 / 6 outside the vein on one side of the edge, that is, 1%;

[0040] Grade 2: 1 - 3%; Lesions appear at 1 / 2 outside the vein on one side of the edge, that is, 3%;

[0041] Grade 3: 4 - 5%; Streaks appear outside the vein on one side of the edge, that is, 4 - 5%;

[0042] Grade 4: 11 - 15%; Streaks appear outside the veins on both sides of the edge, that is, 12%; One streak between the veins, that is, 12.5% (1 / 8);

[0043] Grade 5: 16 - 25%; Two streaks between the veins, that is, 25%

[0044] Grade 6: 26 - 50%; Four streaks between the veins, that is, 50%

[0045] Grade 7: 51 - 75%; Six streaks between the veins, that is, 75%

[0046] Grade 8: 76 - 100%; Seven to eight streaks between the veins, that is, 75% - 100%

[0047] Disease index (%) = Σ (grade value × number of leaves) / (8 × total number of leaves) × 100

[0048] Control effect = (control disease index - treatment disease index) / control disease index × 100

[0049] The control effect of the inoculation test showed that 4 days after inoculating with Xanthomonas oryzae pv. oryzae Xoo, water - soaked leaves began to appear in the control CK treatment group, and no water - soaked leaves appeared in the medicament treatment group. In the following days, the diseased leaves turned into light yellow streaks with wavy edges, and then into yellow streaks. 8 days after inoculation, typical symptoms appeared successively in different treatments, and the control treatment group showed obvious and more serious disease. The disease index and control effect were investigated 14 days after inoculation. The disease index of the FY - 19 bacterial liquid treatment group was 9.2, the disease index of the control chemical agent thiazole zinc treatment group was 0.8, while the disease index of the negative control group reached 32.7. The preventive effect of the FY - 19 bacterial liquid was 72%. The specific statistical results are shown in Table 2 below. 25 days later, most of the rice leaves in the control negative group withered, while individual rice plants sprayed with the biocontrol bacteria showed mild yellowing of the leaf veins.

[0050] Table 2 Control effect of FY - 19 strain on rice bacterial blight in greenhouse pot bioassay

[0051]

[0052]

[0053] The above content of the present invention describes the basic technical solution, technical effect and specific content of implementation of the present invention preferably. The above embodiments are only descriptions of the preferred implementation modes of the present invention. The present invention is not limited by the above embodiments. Without departing from the spirit and scope of the present invention, various changes and improvements made by those of ordinary skill in the art to the technical solution of the present invention shall fall within the protection scope determined by the present invention.

Claims

1. A strain, which is deposited in the General Microbiology Center of the China Committee for Culture Collection of Microorganisms, with the address at No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, and the deposit number is CGMCC NO. 25493, and the deposit date is August 5, 2022. The strain belongs to the species Pseudomonas wayambapalatensis of the Northwest Province, and the strain is named FY-19.

2. Use of the strain according to claim 1, characterized in that The strain is used for preventing and controlling rice bacterial blight or inhibiting the growth of Xanthomonas oryzae pv. Oryzae.

3. The use according to claim 2, wherein The rice bacterial blight is caused by Xanthomonas oryzae pv. Oryzae (Xoo), and the Xanthomonas oryzae pv. Oryzae is the causative agent of rice bacterial blight.

4. An agricultural biocontrol agent, characterized in that, The biocontrol agent contains the cells of the strain described in claim 1.

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