Chromobacterium violaceum and application thereof to prevention and treatment of cotton diseases

Abstract

The application provides a purple-blue-like bacterium and its application in prevention and treatment of cotton diseases, wherein the preservation number of the purple-blue-like bacterium is CCTCC NO: M 20242093. In another aspect, the application provides a use of the CEF642N strain of the purple-blue-like bacterium, which is used as a biological prevention agent for cotton verticillium wilt. The application analyzes the inhibiting effect of the extract of the purple-blue-like bacterium CEF642N on Lecanicillium attenulatum, finds that the extract of the purple-blue-like bacterium CEF642N has the effect of preventing and treating the verticillium wilt, and can be used for preparing a biological prevention agent, thereby providing a new biological prevention method for the prevention and treatment of the verticillium wilt.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of biological control, and particularly relates to a Talaromyces purpureofuscus and application thereof to cotton disease control. BACKGROUND

[0002] Verticillium wilt is one of the most destructive fungal diseases in cotton-growing regions worldwide, causing severe yield loss and fiber quality decline. Its main pathogen is a soil-borne filamentous fungus, which can cause vascular wilting in many important crops. Due to the lack of effective chemical agents to eradicate Verticillium dahliae in the soil, biological control has attracted more and more attention. Currently, the biological control microorganisms for controlling Verticillium wilt mainly include Trichoderma, non-pathogenic Fusarium, Kloeckera, 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 the host. These microorganisms produce antibiotics, enzymes and volatile compounds to protect their host plants from other bacterial and fungal pathogens. Endophytic fungi are the most common microbial preparations used against plant pathogens and are ubiquitous in all plant parts. Natural products and microorganisms have been used as biological pesticides worldwide because they can be obtained from the environment, are generally safe to non-target organisms including humans, have reduced persistence in the environment, and can be used in organic agriculture. Talaromyces is widely distributed in soil, plants, sponges and food. Recent research results show that Talaromyces is very rich in marine environments. Talaromyces secondary metabolites are diverse in structure and have good biological activity, providing a basis for the development and application of endophytic fungi.

[0004] Microbial volatile organic compounds are small molecules from different chemical classes, such as alkenes, alcohols, ketones, organic acids, terpenes, benzenes and pyrazines. Microbial volatile organic compounds can cause many changes in these microorganisms, such as vacuolization, fungal hyphae breakage, loss of intracellular components, regulation of metabolic and pathogenic genes, and expression of important proteins in host responses. Volatile organic compounds are considered a promising, sustainable biological control strategy that can replace pesticides and fertilizers. Mycoparasitism or hyperparasitism is a parasitic interaction between a fungus and another fungus, and is a direct biological control mechanism for controlling plant pathogenic fungi. Fungi can kill plant pathogens, protect plants from abiotic and biotic stress, and reduce disease incidence and severity at the plant population level. SUMMARY

[0005] The purpose of the present application is to provide a Talaromyces purpureofuscus and application thereof to cotton disease control, i.e., Talaromyces purpureofuscus CEF642N and application thereof to cotton Verticillium wilt control.

[0006] The present invention first provides a Talaromyces purpureogenus CEF642N strain, which was deposited in the China Center for Type Culture Collection, 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, which is a biological control agent for cotton Verticillium wilt.

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

[0009] Another aspect of 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 the purple-blue fungus CEF642N to a solvent;

[0010] Furthermore, the preparation contains the extract of CEF642N of the purple-blue fungus at a concentration of 300 μg / mL.

[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) are scanning electron micrographs of cultures grown alone for 15 days at different magnifications; (B1), (B2) and (B3) are scanning electron micrographs of cultures treated with 800 μL / L 1-octen-3-one for 15 days at different magnifications; (C1), (C2) and (C3) are 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 cell structure of Lecanicillium laxum after 15 days of treatment with CEF642N volatile organic compounds: (A) Transmission electron micrograph of L. laxum in the control group; (B) Transmission electron micrograph of L. laxum treated with 800 μL / L 1-octen-3-one; (C) Transmission electron micrograph of L. laxum treated with CEF642N total volatile organic compounds using the double plating method; CW cell wall; M mitochondria; LB liposome.

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

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

[0018] The strain CEF642N of the present application was isolated from the roots of healthy cotton in Anyang, Henan Province. CEF642N was inoculated on PDA medium and a sterile cover glass was inserted beside it. After 10 days, the mycelium was covered with the cover glass, the cover glass was removed, and the conidia were observed under a fluorescence microscope (Nikon, ECLIPSE 80i). CEF642N was cultured on PDA medium for 15 days, cut into 2 cm x 2 cm pieces, and placed on a metal platform coated with conductive glue. Rapid freezing in liquid nitrogen for 1-2 minutes was followed by sublimation and gold spraying for 15 minutes and 1 minute, respectively. The mycelial morphology was observed using a scanning electron microscope (SU3500) in a state filled with liquid nitrogen. According to the ITS sequence of CEF642N, a phylogenetic tree of CEF642N was constructed using the neighbor-joining method with MEGA5 software.

[0019] Confrontation culture: CEF642N (5 mm in diameter) was inoculated on one end of PDA medium, and Vd076 (5 mm in diameter) was inoculated on the other end. The treatment group was 20 mm from the center of the culture dish, and the same diameter was inoculated with Vd076 and CEF642N. The control group was a culture dish inoculated with only Vd076 and CEF642N. After 3 days of culture at 25°C, the colony diameter was measured using the cross method. The data were recorded, the inhibition rate was determined, and the formula was: inhibition rate (%) = [(colony diameter of the control group - 5 mm) - (colony diameter of the treatment group - 5 mm)] / (colony diameter of the control group - 5 mm) x 100.

[0020] In a double-sided culture, CEF642N (5 mm diameter) was inoculated in the center of a dish containing PDA medium, and Vd076 (5 mm diameter) was inoculated in the center of another dish containing PDA medium. The two dishes were then double-sided and sealed with sealing film, with Vd076 on top and CEF642N on the bottom. A culture medium inoculated with Vd076 alone served as a control. Cultures were incubated at 25°C, monitored, and photographed. Colony diameters were measured using the cross-hatch method. Data were collected and the inhibition rate was calculated.

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

[0022] Example 1: Antifungal activity of crude extracts

[0023] The crude extract was dissolved in DMSO and filtered with a 0.22 μm microporous membrane to remove foreign bacteria. 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 were collected, and the inhibition rate was determined. GraphPad Prism 8 was used to calculate the 50% inhibitory 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 exhibited a strong inhibitory effect on Vd076 on day 15 of the confrontation culture, with an inhibition rate of 52.76%. On days 6 and 9 of the confrontation culture, when the antagonist fungus was not in contact with the pathogen, the Vd076 had already grown away from the center. This suggests that CEF642N secretes organic compounds with antifungal activity, which inhibit the growth of Vd076 through diffusion through the agar. In a clasp test, volatile organic compounds produced by CEF642N exhibited a significant inhibitory effect on Vd076 in the absence of any physical contact. CEF642N exhibited a fungal parasitism effect on Vd076 on day 15 of the confrontation culture. This inhibitory effect is attributed to multiple factors, including volatile organic compounds, non-volatile organic compounds, antimicrobial peptides, cell wall-degrading enzymes, and competition for nutrients and space, as CEF642N and Vd076 were present in the same culture dish.

[0025] AntiSMASH was used to predict compound biosynthetic gene clusters in strain CEF642N, identifying 19 gene clusters, including those for polyketide synthases (PKSs), nonribosomal peptide synthetases (NRPSs), and terpenes. Among these, several bioactive compounds with significant anticancer, antibacterial, enzyme inhibitory, and antimalarial activities were discovered. Duklafenamides are dimeric, seven-ring fungal polyketides with significant biological activity. They inhibit adenosine triphosphate (ATP) production through mitorespiration and are effective against several tumor cell lines. Syringic acid is a fascinyltransferase inhibitor with anticancer properties. To validate the genomic predictions, LC-MS / MS was used to analyze the ethyl acetate extract of CEF642N. A total of 6414 compounds were identified, of which 662 were negative. 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 compounds produced by strain CEF642N contribute to antifungal activity, we extracted PDB cultures using ethyl acetate. The results showed that CEF642N extracts inhibited the growth of Vd076 to varying degrees. At 150 μg / mL, CEF642N extract effectively inhibited V. dahliae, causing fungal colonies to shrink and become malformed. At 300 μg / mL, growth of V. dahliae was completely inhibited. Consistent with the counter-culture results, addition of different concentrations of CEF642N crude extract to Oxford cups revealed that the diffusion of non-volatile compounds produced by CEF642N was restricted to a specific area. However, CEF642N continued to grow in the culture dish, consistent with the off-center growth observed in the counter-culture.

[0027] The GC-MS mass spectra were compared with the NIST 2017 database. A total of 411 peaks were detected, and 331 volatile organic compounds were identified. Six of these volatile organic compounds had relative peak areas greater than 0.5. These volatile organic compounds were 1,3-octadiene (retention time Rt 3.371 min), 3-octanone (Rt 11.296 min), 1-octen-3-one (Rt 12.629 min), n-hexanol (Rt 14.247 min), 1-octen-3-ol (Rt 14.247 min), and 2-octen-1-ol (Rt 20.96 min).

[0028] While volatile organic compounds over time, compared with the 6th day group, the 15th day group has 5 volatile organic compounds up-regulated, 46 volatile organic compounds down-regulated, and 280 volatile organic compounds unchanged. There are 63 volatile organic compounds with peak area greater than 0.01, and the clustering heat map analysis shows that three main clusters are found. In cluster 1, the relative volatile organic content of the 6th day group is higher than that of the 15th day group, while in cluster 2 and cluster 3, the relative volatile organic content of the 15th day group is higher. Combined with the analysis of the volcano plot and the volcano cluster heat map of the volatile organic compounds in CEF642N, it is found that among the 331 annotated volatile organic compounds, the relative content of low-level volatile organic compounds is significantly higher in the 6th day than in the 15th day.

[0029] The proportion of main volatile organic compounds changes over time. In the 15th day group, the top three are 1-octen-3-ol (21.87%), 2-octen-1-ol (21.23%) and 3-octanone (20.74%). In the 6th day group, the top 3 are 2-octen-1-ol (28.54%), 1-octen-3-ol (19.19%) and 1-octen-3-ketone (11.81%). The relative percentage of 1-octen-3-ketone in the early stage of culture (6d) is higher than that in the late stage of culture (15d), which may be due to the oxidation of alcohol oxidoreductase to reduce ketone to alcohol. In order to prove their potential biological effect, pure standard samples were purchased and their antagonistic activity against P. lilacina was studied.

[0030] Example 2: Inhibition of P. lilacina by volatile organic compounds produced by P. lilacina

[0031] 1. Inhibition of P. lilacina colony growth

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

[0033] After 15 days of culture on PDA medium, different concentrations of standard samples had inhibitory effect on P. lilacina mycelium. Among them, 1-octen-3-ketone had the highest activity, followed by 2-octen-1-ol and 1-octen-3-ol, and 3-octanone had the lowest activity. EC 50The inhibitory activity of VOCs with different structures may be due to the ketone group being more active than the alcohol group (11-alcohol), as well as differences in the position of the carbon-carbon double bond (13-alcohol) and the position of the carbon-carbon double bond and the alcohol group (E and 11-alcohol). 1-Octen-3-ol had a stronger inhibitory effect than 3-octanone, which is consistent with the inhibitory results against four other pathogenic fungi (Rhizoctonia solani, Fusarium oxysporum, Phytophthora infestans, and Phytophthora citri). Addition of 1 μL of VOCs inhibited the germination of Verticillium dahliae spores. However, the inhibitory effect of 3-octanone was relatively low compared to the other VOCs.

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

[0035] A 10 μL methanol solution containing 1 μL of volatile organic compounds, 90 μL of PDB solution, and 100 μL of spore suspension was added to a 1.8 mL EP tube at a concentration of 1 × 10 6 Spores were cultured in a 200 μL aliquot. The culture was then incubated 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 treated group) / (spore germination rate of the control group) × 100. The germination rates of the control and treated groups were calculated for 24 and 48 hours in the same field of view.

[0036] The main volatile organic compound component of CEF642N, 1-octen-3-one, treated Verticillium dahliae and reduced hyphal development. Among them, one 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 in Verticillium dahliae, thereby reducing the inhibitory effect of external stress. In the buckle 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 showed that the control group was mainly hyphae ( Figure 1 A), while the 1-octen-3-one treated group had some hyphae and spores, and some spores shrank ( 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 affect spore germination, development, and hyphal 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. 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 colonization was consistent with the description of the counterculture of CEF642N and Vd076 described above. CEF642N and Vd076 (5 mm diameter) were plated onto PDA culture medium. Additionally, PDA culture medium coated with Vd076 spores was cultured on three different media for 7 days. CEF642N colony diameters were measured and photographed every three days. CEF642N and Vd076 were cultured counterculture, and the fungal colonies in the countercultures were examined using 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 ​​Vd076 by CEF642N 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 already inoculated with Verticillium dahliae, it was observed that CEF642N could grow on Vd076 hyphae, as well as on medium covered with Vd076 spores. This suggests that CEF642N is capable of bioparasitic growth on Vd076, utilizing nutrients from Vd076 for its own growth. Comparing the colony diameters of CEF642N, it is clear that the largest colony diameter was observed on PDA medium, and there was also a significant difference in colony size between Vd076 hyphae and Vd076 spores. Biotrophic parasitic fungi utilize multiple mechanisms to harvest nutrients from living hosts.

[0042] The scanning electron microscopy images of CEF642N(Tp) cultured alone on PDA medium showed that there were many protrusions on its hyphae ( Figure 4 A), while Vd076 (Vd) mycelium is 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 purple-blue fungus CEF642N for its inhibitory effects on Verticillium dahliae. It inhibits the growth of Verticillium dahliae through the production of volatile organic compounds and non-volatile secondary metabolites, and also exhibits a parasitic effect on the fungus. Genome sequencing analysis revealed that CEF642N contains numerous lipid metabolism genes, which may be precursors to antagonistic volatile organic compounds, as well as genes associated with secondary metabolites such as terpenes and polyketides. Comparative genome analysis revealed that CEF642N is evolutionarily closer to Talaromyces stylosus and possesses a more similar chemical phenotype. Gene deletion and refunctionalization of carbohydrate-active enzymes may have enhanced cell wall degradation capabilities. The mechanism of action of CEF642N against Verticillium dahliae is that volatile organic compounds initially target the pathogen's mitochondria and cell membranes, followed by the production of non-volatile secondary metabolites, ultimately leading to a parasitic effect upon contact. These results suggest that CEF642N possesses multiple antagonistic effects and holds broad application prospects in the prevention and control of cotton Verticillium wilt.

Claims

1. A strain of purple-blue bacteria ( Talaromyces purpureogenus ), characterized in that The preservation number of the purple-blue bacteria is CCTCC NO: M 20242093.

2. Use of the purple-blue fungus according to claim 1 in the preparation of a biological control agent for preventing and controlling cotton Verticillium wilt caused by Verticillium dahliae.

3. The use according to claim 2, characterized in that The biological control agent is a liquid preparation.

4. 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 to prevent and control cotton verticillium wilt caused by Verticillium dahliae.

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