Purple-producing cyanobacteria and application thereof to prevention and treatment of cotton diseases

Abstract

The invention provides a purple blue-like bacterium and an application of the purple blue-like bacterium in prevention and treatment of cotton diseases. The preservation number of the purple blue-like bacterium is CCTCC (China Center For Type Culture Collection) NO: M 20242093. On the other hand, the invention provides an application of the blue purple-producing bacterium CEF642N strain, namely the application of the blue purple-producing bacterium CEF642N strain serving as a biological control agent for cotton verticillium wilt. According to the application disclosed by the invention, the inhibition effect of the purple blue-like bacterium CEF642N extract component on verticillium dahliae is analyzed, and the purple blue-like bacterium CEF642N extract has the effect of preventing and treating the verticillium wilt and can be used for preparing a biological prevention and treatment agent, so that a new biological prevention and treatment means is provided for preventing and treating the verticillium wilt.

Description

Technical Field

[0001] The invention belongs to the technical field of biological control, and particularly relates to a purple-blue fungus and an application thereof in preventing and controlling cotton diseases. Background Art

[0002] Verticillium wilt is one of the most destructive fungal diseases in cotton growing areas around the world, causing severe yield losses and reduced fiber quality. Its main pathogenic agent is a soil-borne filamentous fungus that can cause vascular wilt in a variety of important crops. Due to the lack of effective chemical agents to eradicate Verticillium dahliae in the soil, biological control has attracted increasing attention. At present, biological control microorganisms for the prevention and control of Verticillium wilt are mainly concentrated in Trichoderma, non-pathogenic Fusarium, Vermiculum flavum and Bacillus, Pseudomonas and Streptomyces.

[0003] Endophytic fungi colonize the intercellular and / or intracellular regions of healthy plant tissues and have a close symbiotic relationship with their hosts. These microorganisms produce antibiotics, enzymes, and volatile compounds to protect their host plants from other bacterial and fungal pathogens. Endophytic fungi are used as the most common microbial agents against plant pathogens and are ubiquitous in all plant parts. Natural products and microorganisms have been used as biopesticides worldwide because they can be obtained from the environment, are generally safe for non-target organisms including humans, have reduced persistence in the environment, and they may be used in organic farming. The genus Talaromyces is widely distributed in soil, plants, sponges, and food. Recent research results show that Talaromyces is very abundant in marine environments. The secondary metabolites of Talaromyces are rich in variety, novel in structure, and have good biological activity, which provides a basis for the development and application of endophytes.

[0004] Microbial VOCs are small molecules from different chemical classes such as alkenes, alcohols, ketones, organic acids, terpenes, benzenes, and pyrazines. Microbial VOCs can induce many changes in these microorganisms such as vacuolization, fragmentation of fungal hyphae, loss of intracellular components, modulation of metabolic and pathogenicity genes, and expression of proteins important in host responses. VOCs are considered a promising and sustainable biocontrol strategy that could replace pesticides and fertilizers. Mycoparasitism or hyperparasitism is a parasitic interaction between one fungus and another and is a direct biological control mechanism to control plant pathogenic fungi. Fungi can kill plant pathogens, protect plants from abiotic and biotic stresses, and reduce disease incidence and severity at the plant population level. Summary of the invention

[0005] The purpose of the present invention is to provide a purple-blue bacterium and its application in preventing and controlling cotton diseases, namely, the purple-blue bacterium CEF642N and its application in preventing and controlling cotton Verticillium wilt.

[0006] The present invention first provides a Talaromyces purpureogenus CEF642N strain, which was deposited in the China Type Culture Collection of Wuhan University, Wuhan, China on September 26, 2024, with a deposit number of CCTCCNO: M 20242093.

[0007] In another aspect, the present invention provides a use of the purple-blue fungus CEF642N strain as a biological control agent for cotton Verticillium wilt.

[0008] In yet another aspect, the present invention provides another use of the purple-blue-bacteria-producing CEF642N strain, which is use in preparing a product for inhibiting Verticillium dahliae.

[0009] In another aspect, the present invention provides a liquid preparation for preventing and treating Verticillium wilt in cotton, wherein the preparation is prepared by adding an extract of CEF642N, a purpurogenous fungus, to a solvent;

[0010] Furthermore, the preparation produces a CEF642N extract of 300 μg / mL concentration.

[0011] The present invention also provides a method for preventing and controlling cotton Verticillium wilt, which uses the purple-blue fungus CEF642N as a biological control agent to prevent and control cotton Verticillium wilt;

[0012] Preferably, in the method described above, the concentration of the extract of the purple-blue fungus CEF642N is 300 μg / mL.

[0013] The present invention analyzes the inhibitory effect of the components of the purple-blue fungus CEF642N extract on Verticillium dahliae and finds that the purple-blue fungus CEF642N extract has the effect of preventing and treating Verticillium wilt and can be used to prepare a biological control agent, thereby providing a new biological control means for the prevention and treatment of Verticillium wilt. BRIEF DESCRIPTION OF THE DRAWINGS

[0014] Figure 1 :Effects of volatile organic compounds on the ultrastructure of Verticillium dahliae, (A1), (A2) and (A3) scanning electron micrographs of single culture for 15 days at different magnifications; (B1), (B2) and (B3) scanning electron micrographs of treatment with 800 μL / L 1-octen-3-one for 15 days at different magnifications; (C1), (C2) and (C3) scanning electron micrographs of Verticillium dahliae treated with total volatile organic compounds by CEF642N for 15 days at different magnifications.

[0015] Figure 2: Changes in the intracellular structure of Verticillium dahliae after treatment with CEF642N volatile organic compounds for 15 days: (A) TEM of Verticillium dahliae in the control group; (B) TEM of dahliae treated with 800 μL / L 1-octen-3-one; (C) TEM of Vibrio dahliae treated with CEF642N total volatile organic compounds by double plating method; CW cell wall M mitochondria LB liposomes.

[0016] Figure 3 : Time course of the effects of CEF642N fungal parasitism on Vd076.

[0017] Figure 4 : Scanning electron micrographs of CEF642N (Tp) and Vd076 (Vd) when cultured in confrontation on PDA medium: (A) Scanning electron micrograph of CEF642N cultured alone for 15 days; (B) Scanning electron micrograph of Vd076 cultured alone for 15 days; (C) and (D) Scanning electron micrographs of confrontation cultures for 15 days at different magnifications. DETAILED DESCRIPTION

[0018] The strain CEF642N of the present invention is isolated from healthy cotton roots in Anyang City, Henan Province. CEF642N is inoculated on a PDA culture medium, and a sterile coverslip is inserted obliquely next to it. After 10 days, the mycelium is covered with a coverslip, the coverslip is removed, and the conidia are observed under a fluorescence microscope (Nikon, ECLIPSE 80i). CEF642N is cultured in a PDA culture medium for 15 days, cut into 2 cm×2 cm blocks, and placed on a metal table coated with conductive glue. Quickly freeze in liquid nitrogen for 1-2 minutes, then sublimate and spray gold for 15 minutes and 1 minute respectively. In a state filled with liquid nitrogen, the mycelial morphology is observed using a scanning electron microscope (SU3500). According to the ITS sequence of CEF642N, a phylogenetic tree of CEF642N was constructed using the neighbor connection method using MEGA5 software.

[0019] Confrontation culture: CEF642N (5 mm in diameter) was inoculated at one end of the PDA culture medium, and Vd076 (5 mm in diameter) was inoculated at the other end. The treatment group was inoculated with Vd076 and CEF642N at 20 mm from the center of the culture dish, with the same diameter. The control group was a culture dish inoculated with only Vd076 and CEF642N. Cultured at 25°C for 3 days, the colony diameter was determined by the cross method. Record the data, determine the inhibition rate, and the calculation formula is: Inhibition rate (%) = [(control group colony diameter - 5 mm) - (treatment group colony diameter - 5 mm)] / (control group colony diameter - 5 mm) × 100.

[0020] Cultivation in pairs: CEF642N (5 mm in diameter) was inoculated in the middle of a dish containing PDA medium, and Vd076 (5 mm in diameter) was inoculated in the middle of another dish containing PDA medium. The two dishes were then placed in pairs and sealed with a sealing film, with Vd076 on top and CEF642N on the bottom. The culture medium inoculated with Vd076 alone was used as a control. Culture was performed at 25°C, monitored and photographed. The colony diameter was determined by the cross method. Data were collected and the inhibition rate was calculated.

[0021] The present invention is described in detail below in conjunction with specific embodiments and drawings.

[0022] Example 1: Antifungal activity of crude extracts

[0023] The crude extract was dissolved in DMSO, and the bacteria were removed with a 0.22 μm microporous filter membrane. The final concentrations were 300, 150, 75, 37.5, 18.75 and 0 μg / mL (2% DMSO) in PDA culture medium. The positive control group was treated with carbendazim at a final concentration of 2, 1, 0.5, 0.25, and 0.125 μg / mL. Vd076 was then inoculated in the center of the culture dish. The colony diameter was measured every 3 days, the data was collected, and the inhibition rate was determined. GraphPad Prism 8 was used to calculate the 50% inhibition concentration (EC 50 ).

[0024] A fungal strain isolated from healthy cotton roots in our laboratory has the potential to significantly inhibit the growth of Verticillium dahliae. CEF642N had a strong inhibitory effect on Vd076 on the 15th day of confrontation culture, with an inhibition rate of 52.76%. On the 6th and 9th days of confrontation culture, when the antagonistic fungus did not contact the pathogenic fungus, Verticillium dahliae had grown away from the center, and it was speculated that CEF642N secreted organic matter with antifungal activity, which inhibited the growth of Vd076 through agar diffusion. In the button test, it was found that the volatile organic compounds produced by CEF642N showed a significant inhibitory effect on Vd076 in the absence of any physical contact. CEF642N had a fungal parasitic effect on Vd076 on the 15th day of confrontation culture. This inhibitory effect is caused by multiple reasons, including volatile organic compounds, non-volatile organic compounds, antimicrobial peptides, cell wall degrading enzymes, and nutrient and space competition because CEF642N and Vd076 were present in the same culture dish.

[0025] AntiSMASH was used to predict the biosynthetic gene clusters of compounds in strain CEF642N, and 19 gene clusters were identified, including polyketide synthases (PKS), nonribosomal peptide synthetases (NRPS), and terpenes. Among them, some significant bioactive compounds with anticancer, antibacterial, enzyme inhibitory, and antimalarial activities were found. Dukla penicillins are dimers and seven-ring fungal polyketides with significant biological activities. They inhibit the production of adenosine triphosphate (ATP) through mitotic respiration and effectively inhibit several tumor cell lines. Syringic acid is a fascinyl transferase inhibitor with anticancer effects. To verify the genomic prediction results, LC-MS / MS method was used to detect the ethyl acetate extract of CEF642N. A total of 6414 compounds were found, of which 662 were in negative mode. The most abundant compound was [MH]-m / z 519.1889, followed by m / z 269.104 and m / z 425.1101, respectively.

[0026] To test whether the compounds of strain CEF642N are involved in antifungal activity, we extracted PDB cultures using ethyl acetate as solvent. The results showed that CEF642N extracts inhibited the growth of Vd076 at different levels. CEF642N extracts at 150 μg / mL effectively inhibited V. dahliae, resulting in smaller and deformed fungal colonies. At 300 μg / mL, the growth of V. dahliae was completely inhibited. Consistent with the confrontation culture, addition of different concentrations of CEF642N crude extracts to Oxford cups showed that the diffusion of non-volatile compounds produced by CEF642N was restricted to a specific range. However, CEF642N continued to grow in the culture dish, and V. dahliae showed off-center growth during the confrontation culture, which is consistent with this conclusion.

[0027] The mass spectra of GC-MS were compared with the NIST2017 database. A total of 411 peaks were found and 331 volatile organic compounds were identified. Among them, the relative peak areas of 6 volatile organic compounds were greater than 0.5. These volatile organic compounds were 1,3-octadiene (retention time Rt was 3.371min), 3-octanone (Rt was 11.296min), 1-octen-3-one (Rt was 12.629min), n-hexanol (Rt was 14.247min), 1-octen-3-ol (Rt was 14.247min), and 2-octen-1-ol (Rt was 20.96min).

[0028] As for the volatile organic compounds over time, compared with the 6th day group, 5 volatile organic compounds were up-regulated, 46 volatile organic compounds were down-regulated, and 280 volatile organic compounds remained unchanged in the 15th day group. There were 63 volatile organic compounds with peak areas greater than 0.01, and cluster heat map analysis showed that three major clusters were found. In cluster 1, the relative volatile organic compound content of the 6th day group was higher than that of the 15th day group, while in clusters 2 and 3, the relative volatile organic compound content of the 15th day group was higher. Combining the volcano map of volatile organic compounds in CEF642N and the volcano cluster heat map analysis, it was found that among the 331 annotated volatile organic compounds, the relative content of low-level volatile organic compounds was significantly higher on the 6th day than on the 15th day.

[0029] The proportions of major volatile organic compounds changed over time. In the 15th day group, the top three were 1-octen-3-ol (21.87%), 2-octen-1-ol (21.23%) and 3-octanone (20.74%). In the 6th day group, the top three were 2-octen-1-ol (28.54%), 1-octen-3-ol (19.19%) and 1-octen-3-one (11.81%). The relative percentage of 1-octen-3-one in the early stage of culture (6d) was higher than that in the late stage of culture (15d), which may be due to the reduction of ketone to alcohol catalyzed by alcohol oxidoreductase. In order to demonstrate their potential biological effects, pure standards were purchased and their antagonistic activity against Verticillium dahliae was studied.

[0030] Example 2: Inhibitory effect of volatile organic compounds from purple-blue fungi on Verticillium dahliae

[0031] 1. Inhibitory effect on the growth of Verticillium dahliae colonies

[0032] To determine the effects of volatile compounds on Verticillium dahliae, 20 mL of PDA medium was placed in a 70 mL culture dish. Similar to the confrontation culture, Vd076 was inoculated on one side and an Oxford cup of the same diameter, 20 mm from the center of the culture dish, was placed on the other side. Different volumes of trans-2-octen-1-ol, 1-octen-3-ol, 3-octanone, and 1-octen-3-one were added to the Oxford cup. 1-Octen-3-one was diluted with chromatographic methanol to a concentration of 1.5625-3200 μL / L. The culture conditions and calculation of inhibition rate were the same as before.

[0033] After 15 days of culture on PDA medium, different concentrations of standard products had an inhibitory effect on the hyphae of Verticillium dahliae. Among them, 1-octen-3-one had the highest activity, followed by 2-octen-1-ol and 1-octen-3-ol, while 3-octanone had the lowest activity. 50The values ​​were 14.59, 240.1, 432.9 and 3923 μL / L, respectively. The inhibitory activity of VOCs with different structures may be due to the fact that the ketone group is more active than the alcohol group (11-alcohol), as well as the differences in the positions of the carbon-carbon double bond (13-alcohol) and the carbon-carbon double bond and the alcohol group (E and 11-alcohol). The inhibitory effect of 1-octen-3-ol was stronger than that of 3-octanone, which was consistent with the inhibitory results of the other four pathogenic fungi (Rhizoctonia solani, Fusarium oxysporum, Phytophthora infestans and Phytophthora citri citri). The addition of 1 μL of VOCs inhibited the germination of Verticillium dahliae spores. However, the inhibitory effect of 3-octanone was relatively low compared with other VOCs.

[0034] 2. Inhibitory effect on the germination of conidia of Verticillium dahliae

[0035] Use a 1.8 mL EP tube to add 10 μL of methanol solution containing 1 μL of volatile organic matter, 90 μL of PDB solution, and 100 μL of spore suspension at a concentration of 1×10 6 Spores were cultured in a 200 μL culture system. Then, the culture was carried out at 150 rpm and 25°C. The inhibition rate was calculated as follows: inhibition rate (%) = (spore germination rate of the control group - spore germination rate of the treatment group) / (spore germination rate of the control group) × 100. The germination rates of the control group and the treatment group at 24 h and 48 h in the same field of view were calculated respectively.

[0036] The main volatile organic compound component of CEF642N, 1-octen-3-one, treated Verticillium dahliae and reduced mycelial development. Among them, 1 was treated with 1-octen-3-one at a concentration of 800μL / L. As the treatment time increased, the inhibition rate gradually decreased. This shows that 1-octen-3-one may also cause some reactions of Verticillium dahliae, thereby reducing the inhibitory effect of external stress. In the butt test between CEF642N and Vd076, the total volatile organic compounds produced by CEF642N gradually increased with the extension of treatment time, and the inhibition rate of Verticillium dahliae also gradually increased. Scanning electron microscopy images show that the control group is mainly hyphae ( Figure 1 A), while the 1-octen-3-one treated group had some hyphae and spores, and some spores were atrophied ( Figure 1 B) The volatile organic compound treatment group consisted of spores, which also shrank ( Figure 1 C), indicating that volatile organic compounds hinder spore germination. Total volatile organic compounds and 1-octen-3-one in CEF642N have an effect on spore germination, development and hyphae growth of Verticillium dahliae.

[0037] Transmission electron microscopy results showed that 1-octen-3-one and total volatile organic compounds in CEF642N caused severe organelle damage, mitochondrial degeneration and cavitation compared with the control group. In our study, volatile organic compounds reduced the growth of Verticillium dahliae hyphae, spore germination, and the internal structure of pathogen cells ( Figure 2 ).

[0038] Example 3: Hyperparasitism of the purple-blue fungus CEF642N on Verticillium dahliae Vd076

[0039] The time course of fungal parasitism is consistent with the description of the above-mentioned confrontation culture of CEF642N and Vd076. CEF642N and Vd076 (diameter 5mm) were inoculated into PDA medium. In addition, PDA medium coated with Vd076 spores was cultured on three different media for 7 days. The diameter of CEF642N colonies was measured and photographed every 3 days. CEF642N and Vd076 were cultured in confrontation, and the fungal parasitic part of the confrontation culture was detected by scanning electron microscopy.

[0040] On the 12th day of the confrontation culture, CEF642N and Vd076 came into contact. As the co-culture time increased, the coverage area of ​​CEF642N on Vd076 steadily expanded, indicating that the fungal parasitism rate increased ( Figure 3 ). During the co-culture of CEF642N and Vd076, CEF642N may produce antibiotics to kill pathogens and secrete cell wall-degrading enzymes, including chitinase and β-1,3-glucanase, leading to fungal parasitism.

[0041] When CEF642N was inoculated into a medium that had been inoculated with Verticillium dahliae, it was seen that CEF642N could grow on Vd076 hyphae, as well as on a medium covered with Vd076 spores. This suggests that CEF642N is able to grow biotrophically on Vd076, using nutrients from Verticillium dahliae for its own development. When comparing the colony diameters of CEF642N, it was clear that the PDA medium had the largest colony diameter, and there was also a significant difference in colony size between Vd076 hyphae and Vd076 spores. Biotrophic parasitic fungi use a variety of mechanisms to collect nutrients from living hosts.

[0042] The scanning electron microscopy image of CEF642N(Tp) cultured alone on PDA medium showed that there were many protrusions on its hyphae ( Figure 4 A), while the hyphae of Vd076 (Vd) are smooth ( Figure 4 B) In the parasitic part of the confrontation culture, CEF642N hyphae can grow in parallel with Verticillium dahliae ( Figure 4 C, D).

[0043] In summary, the present invention screened the inhibitory effect of purple-blue fungi CEF642N on Verticillium dahliae. It can inhibit the growth of Verticillium dahliae through volatile organic compounds and non-volatile secondary metabolites, and also has fungal parasitic effects on Verticillium dahliae. Genome sequencing analysis shows that CEF642N contains a large number of lipid metabolism genes, which may be precursors of antagonistic volatile organic compounds, as well as related genes containing secondary metabolites such as terpenes and polyketides. Genome comparative analysis shows that CEF642N is evolutionarily closer to the stalked basket fungus and has a closer chemical phenotype. Carbohydrate-active enzymes may have more cell wall degradation functions after gene deletion and new functionalization. The mechanism of action of CEF642N on Verticillium dahliae is that early volatile organic compounds target the mitochondria and cell membranes of pathogens, followed by non-volatile secondary metabolites, and finally produce parasitic effects after contact. The results show that CEF642N has multiple antagonistic effects and has broad application prospects in the prevention and control of cotton wilt.

Claims

1. A purple-blue-producing bacterium, characterized in that: The preservation number of the purple-blue bacteria is CCTCCNO: M20242093.

2. Use of the purple-blue fungus according to claim 1 as a biological control agent for cotton Verticillium wilt.

3. Use of the purple-blue bacteria according to claim 1 in the preparation of products for inhibiting Verticillium dahliae.

4. The use according to claim 3, characterized in that The product is a liquid preparation.

5. A liquid preparation for preventing and treating Verticillium wilt of cotton, characterized in that: The liquid preparation is prepared by adding the extract of the purple-blue bacteria described in claim 1 into a solvent.

6. The liquid preparation according to claim 5, characterized in that The extract is prepared by extraction using ethyl acetate as solvent.

7. A method for preventing and treating cotton Verticillium wilt, characterized in that: The method is to use the purple-blue fungus described in claim 1 or the liquid preparation described in claim 5 to prevent and control cotton Verticillium wilt.

8. The method according to claim 7, characterized in that The concentration of the extract in the liquid preparation is 300 μg / mL.

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