Bacillus velezensis with antagonism effect on cotton verticillium wilt and high salt stress resistance and application of bacillus velezensis

Abstract

The invention discloses bacillus velezensis with antagonism on cotton verticillium wilt and high salt stress resistance and application, the strain has the advantages of resisting saline and alkaline and antagonizing the cotton verticillium wilt at the same time, the problem of verticillium wilt in the existing saline-alkali soil cotton planting process is solved, the utilization of saline-alkali soil is expanded, and the cotton yield is increased.

Description

(I) Technical field

[0001] The invention belongs to the field of agricultural planting and application of microorganisms, and specifically relates to a Bacillus Velezii which has antagonistic effect on cotton Verticillium wilt and is resistant to high salt stress and an application thereof. (II) Background technology

[0002] Cotton is an important natural fiber crop and the main raw material and strategic material source for the textile industry. Cotton Verticillium wilt is caused by plant pathogenic fungi Verticillium dahliae and V.albo-atrum. The pathogen has strong survival and colonization ability in the soil and can survive in the soil all year round. It is a very stubborn typical soil-borne disease, causing huge economic losses to cotton production. At present, there are no good disease-resistant varieties in the prevention and control of cotton Verticillium wilt. The main measures taken are crop rotation and application of chemical pesticides, but the prevention effect is low.

[0003] Biological control is an effective control strategy for soil-borne diseases. Biological control has attracted attention and has been applied in practice due to its advantages such as environmental protection and sustainable benefits. At present, biological control of soil-borne diseases such as cotton Verticillium wilt mainly uses antagonistic microorganisms, including bacteria, fungi, actinomycetes, such as Pseudomonas fluorescens, Bacillus, Trichoderma harzianum, Streptomyces, etc.

[0004] Bacilus velezensis is distributed in a variety of habitats in nature, is highly safe, and can produce antagonistic or killing effects on a variety of plant pathogenic fungi and pests (such as mites). Bai Hongyan et al. (2022) used confrontation culture to find that the EBV02 strain of Bacilus velezensis has an inhibitory effect on the mycelial growth of Verticillium dahliae Vd080. Zhao Hao (2023) found that Bacillus velezensis LJ02 has a strong antagonistic effect on Verticillium dahliae VD592, which can inhibit the growth of Verticillium dahliae and destroy the mycelial structure. Zhang Qiong et al. (2020) found that the SZAD1 strain of Bacillus velezensis can significantly inhibit the mycelial growth of VD080 and has the potential to be used as a biocontrol agent to control cotton Verticillium wilt. Pan Wen et al. (2024) screened out myxobacteria with good antagonism to Verticillium dahliae.

[0005] Patent applications published in recent years also show related strains and technologies for the prevention and treatment of cotton Verticillium wilt using antagonistic bacteria. CN118325789A developed a composite microbial agent including Penicillium simplicisum, Trichoderma viride, Bacillus licheniformis, Bacillus amyloliquefaciens and Bacillus velez for the prevention and treatment of cotton Verticillium wilt; CN117362108A provides a cotton flowering and boll period special fertilizer including Trichoderma and Bacillus velez, which also has a certain preventive effect on Verticillium wilt; CN115191443A provides a microbial agent including Pseudomonas alcaligenes, Bacillus velez and Burkholderia, which can reduce the incidence of cotton Verticillium wilt.

[0006] Cotton is drought-resistant and salt-tolerant and can be grown as a pioneer crop on saline-alkali land. 2 Saline-alkali land has been developed for cotton planting, and China has become the country with the largest scale of cotton planting in saline-alkali land in the world. It is also facing the problem of prevention and control of Verticillium wilt (Xue Zhizhong, 2011). However, the development of antagonistic bacteria resources for the prevention and control of Verticillium wilt in cotton on saline-alkali land is not enough. It is also necessary to screen strains with strong salt tolerance and excellent antagonism to Verticillium wilt. Patent application CN116286500A reported that a plant endogenous salt-tolerant Bacillus Velez was screened from Xinjiang halophytes. The strain can grow in the solid culture medium of Tryptose soya agar (TSA) with sodium chloride (30g / L); CN117305146A provides a marine-derived Bacillus Velez LG-13 with obvious anti-Aspergillus niger effect, which can grow in a culture medium with an addition of NaCl of 4-6g / L. In August 2024, it was reported that the Hebei Academy of Agriculture and Forestry Sciences used salt-alkali resistant bacteria to apply to severely saline-alkali cotton fields in Xinjiang, revitalizing the saline-alkali land (DOI:10.28603 / n.cnki.nnmrb.2024.004074.2024-08-10(006)).

[0007] Xinjiang is the largest cotton producing area in my country, and its cotton output accounts for more than 85% of the country's total output. However, saline-alkali land is an important factor restricting Xinjiang's cotton production. Coupled with compound factors such as Verticillium wilt, the reduction in production has increased. Cultivating and screening cotton germplasm that is resistant to Verticillium wilt and salt-tolerant is a better strategy in the long run (Ma Qingqian, 2023), but crop breeding is slow to take effect. The area of ​​saline-alkali land in Changji Prefecture, Xinjiang is 22422.96ha (Wang Zhen et al. 2024), which shows that the task of improving saline-alkali land is arduous on the one hand, and on the other hand, it contains huge salt-alkali-tolerant microbial resources. Therefore, screening and applying microbial resources that can inhibit Verticillium wilt and tolerate salt have great practical application significance. (III) Summary of the invention

[0008] The present invention aims to provide a Bacillus Velezii strain which has an antagonistic effect on cotton Verticillium wilt and is resistant to high salt stress and its application. The strain has the advantages of being salt-tolerant and antagonistic to cotton Verticillium wilt, thereby solving the problem of Verticillium wilt occurring during the existing cotton planting process in saline-alkali land, expanding the utilization of saline-alkali land and increasing cotton yield.

[0009] The technical solution adopted by the present invention is:

[0010] The present invention provides a Bacillus velezensis KYF24-01C which has an antagonistic effect on cotton Verticillium wilt and is resistant to high salt stress, and is deposited in the General Microbiological Center of China Microorganism Culture Collection Committee with a deposit number of CGMCC NO.32365 and a deposit date of October 28, 2024. The address is: No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, 100101.

[0011] The present invention also provides an application of the Bacillus Velezii KYF24-01C in preparing an antagonistic bacterial agent against Verticillium wilt of cotton in high saline-alkali soil.

[0012] Furthermore, the antagonistic bacterial agent is a bacterial solution obtained by expanding the culture of Bacillus velezii KYF24-01C.

[0013] Furthermore, the bacterial liquid is prepared by inoculating Bacillus Velezii KYF24-01C into LB solid culture medium containing 5-10 g / L NaCl, culturing at 37° C. for 1-2 days; and then inoculating into LB liquid culture medium, culturing at 37° C. and 180 rpm for 1-2 days.

[0014] Compared with the prior art, the beneficial effects of the present invention are mainly reflected in:

[0015] The invention provides a strain of Bacillus velezii KYF24-01C, which has a strong inhibitory effect on cotton Verticillium wilt, and the inhibition rate reaches 83.46% under the condition of plate confrontation culture. Moreover, the strain can tolerate high salt of 5% NaCl concentration.

[0016] Bacillus Velez KYF24-01C has good application potential in the prevention and control of Verticillium wilt in cotton, especially in the prevention and control of Verticillium wilt in cotton grown in saline-alkali land, and provides a reference for expanding the comprehensive utilization of saline-alkali land. (IV) Description of the drawings

[0017] Figure 1 , 16S rDNA sequence BLAST analysis (F primer).

[0018] Figure 2 , 16S rDNA sequence BLAST analysis (R primer).

[0019] Figure 3 , Colony photos of Bacillus velez KYF24-01C on high salt (5% NaCl) medium.

[0020] Figure 4 , photos of colonies of the antagonistic culture of Bacillus Velezii KYF24-01C and Verticillium dahliae of cotton; A antagonistic culture; B cotton Verticillium dahliae control. (V) Specific implementation methods

[0021] The present invention is further described below in conjunction with specific embodiments, but the protection scope of the present invention is not limited thereto:

[0022] Example 1. Isolation and identification of strain KYF24-01C

[0023] 1. Isolation of strains

[0024] (1) Sampling

[0025] Soil samples were collected from saline-alkali land in Ruoqiang County, Bayinguoleng Mongol Autonomous Prefecture, Xinjiang Uygur Autonomous Region, and brought back to the laboratory for microbial isolation.

[0026] (2) Salt tolerance determination and screening

[0027] Microbial isolation was carried out by the dilution plate method. Single microbial colonies of different forms were isolated on high-salt PDA, LB and YPD culture media. The specific method is as follows:

[0028] Take a 50mL beaker, add 9mL sterile water, weigh 1g soil sample, add it to the beaker and stir with a glass rod for 30min. After standing for 3min, take 100μL supernatant and dilute it 1000 times with sterile water, and spread it on high-salt PDA medium, high-salt LB medium and high-salt YPD medium plates respectively. The high-salt PDA medium plate is placed in a 25℃ biochemical incubator for 2-3 days, the high-salt YPD medium plate is placed in a 30℃ biochemical incubator for 1-2 days, and the high-salt LB medium plate is placed in a 37℃ biochemical incubator for 1-2 days. Each medium plate can be stopped in time according to the appearance of single colonies.

[0029] High-salt PDA culture medium: 200 g potatoes (peeled and cut into small pieces, boiled and filtered through gauze to obtain the filtrate), 20 g glucose, 30 g NaCl, 15 g agar, and deionized water to 1 L.

[0030] High-salt LB medium: peptone 10 g, yeast extract 5 g, NaCl 30 g, agar 15 g, and deionized water to 1 L.

[0031] High-salt YPD medium: 10 g yeast extract, 20 g peptone, 20 g glucose, 30 g NaCl, 15 g agar, and deionized water to 1 L.

[0032] The above culture media were sterilized at 121°C for 15 min.

[0033] (3) Antagonistic effect determination and screening

[0034] After obtaining single colonies by culturing on the above-mentioned high-salt culture medium, colonies with obviously different characteristics such as colony morphology and color are selected for numbering and marking, and single colonies with different marking numbers are picked out for antagonism test against cotton Verticillium wilt.

[0035] Candidate antagonistic bacteria to be tested: The selected single colony was inoculated into LB medium plate, activated and cultured at 37°C for 1-2 days, and then inoculated into LB liquid culture medium, cultured at 37°C and 180 rpm for 1 day, and the obtained culture solution was used as the antagonistic bacteria to be tested.

[0036] Cultivation of cotton Verticillium wilt pathogen: Verticillium dahliae was provided by the Institute of Plant Protection, Xinjiang Academy of Agricultural Sciences and inoculated on a PDA medium (200 g of boiled potato juice, 20 g of glucose, 15 g of agar, and deionized water to make up to 1 L) plate. After culturing at 28°C for 7 days, holes were punched in the edge area of ​​the colony to obtain bacterial cakes (2 cm in diameter).

[0037] Antagonistic effect detection: Inoculate the cotton Verticillium wilt fungus cake in the center of a fresh PDA culture medium plate, culture at 28°C for 3 days, draw a short line (length 2-3 cm) about 1.5-2 cm from the edge of the culture dish to inoculate the antagonistic bacteria to be tested, continue to culture at 28°C, and observe the antagonistic effect of the bacteria to be tested. According to the antagonistic results, a strain with strong salt tolerance and good antagonistic effect was selected, recorded as strain KYF24-01C.

[0038] 2. Identification of strain KYF24-01C

[0039] The strain KYF24-01C was streaked continuously on LB medium plates and cultured at 37°C for 1 day. A single colony (pure culture) was picked and sent to a DNA sequencing commercial company (Qingke Biotechnology Co., Ltd.) for 16S rDNA sequencing. The nucleotide sequence obtained by the F primer is shown in SEQ ID NO.1, and the nucleotide sequence obtained by the R primer is shown in SEQ ID NO.2. The obtained sequence was compared and analyzed by BLAST (Basic Local Alignment Search Tool) in GenBank, and the results are shown in Figure 1 and Figure 2 The possible bacterial species were preliminarily identified based on sequence similarity.

[0040] After BLAST analysis of 16S rDNA sequence, Qingke Biotech provided dual primer sequencing results. The sequencing results of the two primers were analyzed by BLAST sequence comparison results, showing that the bacteria with the highest similarity were both Bacillus velezensis. The strain KYF24-01C was identified as Bacillus velezensis and named as Bacillus velezensis KYF24-01C. It was deposited in the General Microbiology Center of China National Culture Collection Administration with the deposit number CGMCC NO.32365.

[0041] F primer sequence: AGTTTGATCMTGGCTCAG

[0042] R primer sequence: GGTTACCTTGTTACGACTT

[0043] F primer sequencing results (SEQ ID NO.1):

[0044] AGTCCTTAATGATGACTGTGAAGTGTTAGGGGTTTTCCGTCCCTTAGTGCTGCTATAACGCGTTAATGACTCCGTGTGGAGAGTACGGGCGCAAGACTGAATCTCAAAGGAATTGACGGACCCCCGCACAAGCGGGGTAATGTGTGTTTTATTTCTAAGCAACGCAAAAAATCTTACCAGGTTTTGACGTCCTCTGACTATCCTAGAGATATGACGGCCCCTTCGAGAACATAGTGACAGGTGCTGCATGGTTGTCGTCAGCTCGTGTCGTGAGATGTTGGGTTAAGTCCCGCAACGAGCGCAACCCTTGATCTTAGTTGCCACATTTCAGTTGGGCACTCTAAGGTGACTGCCGGGGACAAACCGAAGGAAGGGGGGAATGACGTCAAATCATCATGCCCCTTATGACCTGGGTTACCCCCGTGCTACAAGGGAGAGAACAAAGGGCACCGAACCCGCAAGGTTAAGTCAATCCCACAAATCTGTTCTCAGCTCGAATCGCAGCCTGCAACTCAACTGCGGGAAGCTGAAATCGTTAGAAATCGCGAATCAGCTTGCCGGGGGGAATACGTTCCCGGGCCTTGAACACCCCGCCCGCCACACCTGGAAAGTTTGTAACACCCGAAGACGGAGACGAAACCTTTGAAGAGCCATTCGCCGAAGGTGGGACAGATGAGTGGTGAGATCCGAGGGGGGAAAACCCCCAAAA

[0045] Sequencing result of primer R (SEQ ID NO.2):

[0046] AGCCCAATACTCTGCGACCGTACTCCCCAGGCGGAGTGCTTCATGCGTTAGCTGCAGCACTAAGGAAAGTAAAACCCCCTAACCTTATGACTCATCGTTTACGGCGTGCTATACCAGGATATCTAATCGTGTTCGCTCCCCACTTTTTCGCTCCTCAGCGACAACTACAGACCAGAAAGGCCTCTTCCCCACTGGTGTTCCTCCACATCTCTACGTATTTCTCCGATACGCGTGAAATTCCACTCTCCTCTTGTGCACTCAAGTTCCCCAGTTTCCAGTGACCCTCCCCGGGTGACCCGGGGGTTTTCACATGACATTTATAAACCCGTCTGCGAGCCCTTTACCCCCAAAAATTCCGGACAACGCTTGCCACCTACGTATTACCGCGTGTGGTGACACGGATATACGCGGGGTTTTCTGACTAGGCACCGATGAGGCGCCGGCCTATTTGAACGGTACTTGTTCTTCCCTAAAAACAGAGTTTTACGATCCGAAAACCTTCCTCACTCACGCGTTGTTGCTCCGGCATATTTTCCACTATTGAAAAAGATTCCCTACTGCTGCCTCCAGTAGGAGGCTGGGCCGTGCCTCACCCCCAGGGTGGCCGATCACCCTCTCACGTCTACTACGCATCGGCTTGTTGATGAGCCATTACCTCACCAACTAGATAATGCATCGCGCGCTCATCTGTAACTGGAACCCCGAAGCCACCTGTTTATGTCTGAACCATGCGATGCAGACAAACATACCGCTATTAAGCCCGTATCCCCCGGAGACTATCCCAGTCTTACATGCATTACTACACCACGTGCTACTCACGCCGTCCGTCCGCTAAACATCGGTGACGAAGCTCCTCATCTGTCCGCTCGACGTCGACGATAGTAGCCCCCCGCGCCCAAGCTTTCTCTTTGGCGAGAAAAAAAAA

[0047] Example 2. Determination of the high-salt tolerance of Bacillus velezensis KYF24-01C

[0048] Bacillus Velezii KYF24-01C was streaked onto LB medium plates containing 5 g / L NaCl and cultured at 37°C for 1 day. The colony photos are shown in Figure 3 The results showed that Bacillus velezii KYF24-01C could grow well on LB medium with a NaCl content of 5 g / L, which proved that the strain had the characteristics of high salt tolerance.

[0049] Example 3: Antagonistic effect of Bacillus velezensis KYF24-01C on cotton Verticillium dahliae

[0050] Antagonistic bacteria culture: Bacillus velezensis KYF24-01C was inoculated into LB medium containing 10 g / L NaCl and activated and cultured at 37°C for 1-2 days.

[0051] Cultivation of cotton Verticillium wilt pathogen: Verticillium dahliae was provided by the Institute of Plant Protection, Xinjiang Academy of Agricultural Sciences and inoculated on a PDA medium plate. After culturing at 28°C for 7 days, a hole was punched at the edge of the colony to obtain a bacterial cake (2 cm in diameter).

[0052] Antagonistic effect test: Inoculate the Verticillium dahliae cake in the center of a fresh PDA culture medium plate, culture at 28°C for 3 days, draw a short line (length 2-3 cm) about 1.5-2 cm from the edge of the culture dish to inoculate the antagonistic bacteria, and continue to culture at 28°C for 7-10 days. Figure 4 A. The control group was not inoculated with antagonists. The colony photos are shown in Figure 4 Middle B.

[0053] The results showed that after 7-10 days of confrontation culture, the edge of the Verticillium wilt colony of cotton grew to about 1.5 cm away from Bacillus velezii KYF24-01C, and the growth of Verticillium wilt was significantly inhibited. The edge of the colony was dense, the colony color was brown, and the colony no longer expanded, while the control strain colony could continue to grow until the entire plate was full. The results showed that KYF24-01C had a significant antagonistic effect on Verticillium wilt of cotton. The colony area was determined by the cross method, and the antibacterial rate was calculated to be 83.46%.

[0054] Inhibition rate (%) = 1-diameter of treated pathogens ÷ diameter of control pathogens × 100.

Claims

1. A strain of Bacillus velezensis KYF24-01C that is antagonistic to cotton Verticillium wilt and resistant to high salt stress is deposited in the General Microbiology Center of China Microorganism Culture Collection Administration, with the deposit number CGMCC NO.32365 and the deposit date October 28, 2024. The address is No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, 100101.

2. Use of the Bacillus Velezii KYF24-01C according to claim 1 in preparing an antagonistic agent against Verticillium dahliae in high saline-alkali soil.

3. The use according to claim 2, characterized in that The antagonistic bacteria is a bacterial liquid obtained by expanding the culture of Bacillus Velezii KYF24-01C.

4. The use according to claim 3, characterized in that The bacterial liquid is prepared by inoculating Bacillus Velezii KYF24-01C into LB solid culture medium containing 5-10 g / L NaCl, culturing at 37° C. for 1-2 days; and then inoculating into LB liquid culture medium, culturing at 37° C. and 180 rpm for 1-2 days.

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