Talaromyces purpureogenus and use thereof in prevention and control of cotton disease

Abstract

Provided in the present invention are Talaromyces purpureogenus and use thereof in the prevention and control of a cotton disease. The deposit number of the Talaromyces purpureogenus is CCTCC NO: M 20242093. A further aspect of the present invention provides use of a Talaromyces purpureogenus CEF642N strain, which is used as a biological control agent for cotton Verticillium wilt. In the present invention, the inhibitory effect of a Talaromyces purpureogenus CEF642N extract component on Verticillium dahliae is analyzed, and it is found that the Talaromyces purpureogenus CEF642N extract has the effect of preventing and controlling Verticillium wilt, and can be used for preparing a biological control agent, thereby providing a new biological control means for the prevention and control of Verticillium wilt.

Description

A purple-blue shaped fungus and its application in controlling cotton diseases. Technical Field This invention belongs to the field of biological control technology, specifically relating to a purple-blue morphological fungus and its application in controlling cotton diseases. Background Technology Verticillium wilt is one of the most destructive fungal diseases in cotton-growing areas worldwide, causing severe yield losses and decreased fiber quality. Its main causative agent is a soil-borne filamentous fungus that can cause vascular bundle wilting in a variety of important crops. Due to the lack of effective chemical agents to eradicate Verticillium dahliae in the soil, biological control is receiving increasing attention. Currently, the main biological control microorganisms for Verticillium wilt include Trichoderma, non-pathogenic Fusarium, Demodex flavus, Bacillus, Pseudomonas, and Streptomyces. Endophytic fungi colonize the intercellular and / or intracellular regions of healthy plant tissues, maintaining 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 worldwide as biopesticides because they are available from the environment, are generally safe for non-target organisms including humans, have low persistence in the environment, and may be suitable for organic agriculture. Bassilago farfara (basiliformes) are widely distributed in soil, plants, sponges, and food. Recent studies have shown that Bassilago farfara is very abundant in marine environments. The diverse and novel secondary metabolites of Bassilago farfara possess excellent biological activity, providing a foundation for the development and application of endophytic fungi. Microbial volatile organic compounds (VOCs) are small molecules from various chemical classes, such as alkenes, alcohols, ketones, organic acids, terpenes, benzenes, and pyrazines. VOCs can induce numerous changes in these microorganisms, such as cavitation, fungal hyphal fragmentation, loss of intracellular components, regulation of metabolism and pathogenic genes, and expression of proteins important in host responses. VOCs are considered a promising and sustainable biocontrol strategy that could replace pesticides and fertilizers. Fungal parasitism, or hyperparasitism, is a parasitic interaction between fungi and another fungus, representing a direct biological control mechanism for 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 The purpose of this invention is to provide a purple-blue morphophyte and its application in the control of cotton diseases, namely, the purple-blue morphophyte CEF642N and its application in the control of cotton Verticillium wilt. The present invention first provides a strain of purpureogenus (Talaromyces purpureogenus) CEF642N, which was deposited on September 26, 2024, at the China Center for Type Culture Collection, Wuhan University, China, with accession number CCTCC NO:M 20242093. In another aspect, the present invention provides an application of the aforementioned purple-blue bacterium strain CEF642N as a biological control agent for cotton Verticillium wilt. In another aspect, the present invention also provides another use of the aforementioned purple-blue bacterium strain CEF642N, in the preparation of products for inhibiting Verticillium dahliae. In another aspect, the present invention provides a liquid preparation for controlling cotton Verticillium wilt, wherein the preparation is prepared by adding an extract of CEF642N of *Cymbidium purpureum* to a solvent. Furthermore, the extract of *Cymbidium violaceum* CEF642N from the formulation is at a concentration of 300 μg / mL. The present invention also provides a method for controlling cotton Verticillium wilt, which uses CEF642N, a bacterium that produces purple-blue spores, as a biological control agent to control cotton Verticillium wilt; Preferably, in the method, the concentration of the extract of CEF642N of the purple-blue bacterium is 300 ug / mL. This invention analyzed the inhibitory effect of CEF642N extract of *Verticillium purpureum* on *Verticillium dahliae*, and found that the extract of CEF642N has the effect of preventing and controlling Verticillium wilt, and can be used to prepare biocontrol agents, thus providing a new biological control method for the prevention and control of Verticillium wilt. Attached Figure Description Figure 1: Effects of volatile organic compounds on the ultrastructure of Verticillium dahliae. SEM images of (A1), (A2), and (A3) cultured alone for 15 days at different magnifications; SEM images of (B1), (B2), and (B3) treated with 800 μL / L1-octen-3 one for 15 days at different magnifications; SEM images of (C1), (C2), and (C3) of Verticillium dahliae treated with CEF642N total volatile organic compounds for 15 days at different magnifications. Figure 2: Changes in the intracellular structure of Verticillium dahliae after 15 days of treatment with CEF642N volatile organic compounds: (A) Transmission electron micrograph of Verticillium dahliae in the control group; (B) Transmission electron micrograph of Verticillium dahliae treated with 800 μL / L 1-octen-3 one; (C) Transmission electron micrograph of Vibrio dahliae treated with CEF642N total volatile organic compounds using the double plating method; CW cell wall, M mitochondria, LB liposomes. Figure 3: The time course of the effect of CEF642N fungal parasitism on Vd076. Figure 4: Scanning electron microscope (SEM) images of CEF642N(Tp) and Vd076(Vd) in confrontation culture on PDA medium: (A) SEM image of CEF642N cultured alone for 15 days; (B) SEM image of Vd076 cultured alone for 15 days; (C) and (D) SEM images of confrontation cultured for 15 days at different magnifications. Detailed Implementation The strain CEF642N of this invention was isolated from the roots of healthy cotton plants in Anyang City, Henan Province. CEF642N was inoculated onto PDA medium, with a sterile coverslip inserted obliquely beside it. After 10 days, the hyphae were covered with the coverslip, which was then removed, and the conidia were observed under a fluorescence microscope (Nikon, ECLIPSE 80i). CEF642N was cultured in PDA medium for 15 days, cut into 2cm x 2cm pieces, and placed on a metal stage coated with conductive adhesive. The pieces were rapidly frozen in liquid nitrogen for 1-2 minutes, followed by sublimation and gold spraying for 15 minutes and 1 minute, respectively. Hyphae morphology was observed under liquid nitrogen using a scanning electron microscope (SU3500). Based on the ITS sequence of CEF642N, a phylogenetic tree of CEF642N was constructed using the neighbor-joining method with MEGA5 software. Confrontation culture: CEF642N (5 mm in diameter) was inoculated at one end of PDA medium, and Vd076 (5 mm in diameter) was inoculated at the other end. Treatment groups were inoculated with both Vd076 and CEF642N at a distance of 20 mm from the center of the culture dish, with the same diameter. The control group consisted of culture dishes inoculated only with Vd076 and CEF642N. The cultures were incubated at 25°C for 3 days, and the colony diameter was measured using the cross-sectional method. Data were recorded, and the inhibition rate was determined using the formula: Inhibition rate (%) = [(Control group colony diameter - 5 mm) - (Treatment group colony diameter - 5 mm)] / (Control group colony diameter - 5 mm) x 100. Inoculation method: CEF642N (5 mm in diameter) was inoculated in the middle of a petri dish containing PDA medium, and Vd076 (5 mm in diameter) was inoculated in the middle of another petri dish containing PDA medium. The two petri dishes were then inoculated together and sealed with sealing film, with Vd076 on top and CEF642N on the bottom. A medium inoculated solely with Vd076 served as a control. Incubation was carried out at 25°C, and colony diameter was monitored and photographed. The cross-sectional method was used to determine the colony diameter. Data were collected, and the inhibition rate was calculated. The present invention will now be described in detail with reference to specific embodiments and accompanying drawings. Example 1: Antifungal activity of crude extract The crude extract was dissolved in DMSO, and contaminants were removed using a 0.22 μm microporous membrane. The final concentrations were 300, 150, 75, 37.5, 18.75, and 0 μg / mL (2% DMSO) in PDA medium. The positive control group was treated with carbendazim at final concentrations of 2, 1, 0.5, 0.25, and 0.125 μg / mL. Vd076 was then inoculated into the center of each culture dish. Colony diameter was measured every 3 days, data were collected, and the inhibition rate was determined. A GraphPad Prism 8 was used to calculate the 50% inhibitory concentration (EC50). A fungal strain isolated from healthy cotton roots in our laboratory has the potential to significantly inhibit the growth of *Verticillium dahliae*. CEF642N showed a strong inhibitory effect on Vd076 on day 15 of confrontation culture, with an inhibition rate of 52.76%. On days 6 and 9 of confrontation culture, when the antagonistic fungus was not in contact with the pathogenic fungus, *Verticillium dahliae* had already deviated from the center of growth, suggesting that CEF642N secretes antifungal organic matter, which inhibits the growth of Vd076 through agar diffusion. In the inversion experiment, it was found that the volatile organic compounds produced by CEF642N showed a significant inhibitory effect on Vd076 without any physical contact. CEF642N also showed a fungal parasitic effect on Vd076 on day 15 of confrontation culture. This inhibitory effect is caused by multiple factors, including volatile organic compounds, non-volatile organic compounds, antimicrobial peptides, cell wall degrading enzymes, and nutrient and space competition, as CEF642N and Vd076 were present in the same culture dish. Using AntiSMASH to predict the biosynthetic gene clusters of compounds in strain CEF642N, 19 gene clusters were identified, including polyketide synthase (PKS), nonribosomal peptide synthase (NRPS), and terpenes. Among these, several compounds with significant bioactive activities, including anticancer, antibacterial, enzyme inhibition, and antimalarial activity, were discovered. Duclapenicillin, a dimer and heptacyclic fungal polyketide, exhibits significant bioactivity. It inhibits adenosine triphosphate (ATP) production through mitotic respiration, effectively suppressing several tumor cell lines. Syringic acid, an alkyne transferase inhibitor, possesses anticancer activity. To validate the genome prediction results, we used LC-MS / MS to detect 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. To investigate whether the compounds from strain CEF642N were related to antifungal activity, we extracted PDB cultures using ethyl acetate as the solvent. The results showed that the CEF642N extract inhibited the growth of Vd076 to varying degrees. At 150 μg / mL, the CEF642N extract effectively inhibited *Verticillium dahliae*, leading to smaller and deformed fungal colonies. At 300 μg / mL, *Verticillium dahliae* growth was completely inhibited. Consistent with confrontation cultures, different concentrations of crude CEF642N extract were added to Oxford cups, and the results indicated that the diffusion of non-volatile compounds produced by CEF642N was confined to a specific area. However, CEF642N continued to grow in the culture dishes, and during confrontation cultures, *Verticillium dahliae* exhibited off-center growth, consistent with this conclusion. The GC-MS mass spectra were compared with the NIST 2017 database. A total of 411 peaks were found, and 331 volatile organic compounds (VOCs) were identified. Among them, 6 VOCs had a relative peak area greater than 0.5. These VOCs 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] Over time, compared to the day 6 group, the day 15 group saw 5 volatile organic compounds (VOCs) upregulated, 46 downregulated, and 280 unchanged. 63 VOCs had peak areas greater than 0.01. Cluster heatmap analysis revealed three main clusters. In cluster 1, the relative VOC content of the day 6 group was higher than that of the day 15 group, while in clusters 2 and 3, the relative VOC content of the day 15 group was higher. Analysis combining the volcano map and volcano swarm heatmap of VOCs in CEF642N revealed that among the 331 annotated VOCs, the relative content of low-level VOCs was significantly higher on day 6 than on day 15. The proportions of major volatile organic compounds varied over time. In the day 15 group, the top three were 1-octen-3-ol (21.87%), 2-octen-1-ol (21.23%), and 3-octenone (20.74%). In the day 6 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 was higher in the early stage of culture (6 days) than in the later stage (15 days), which may be due to the reduction of ketones to alcohols catalyzed by alcohol oxidoreductase. To demonstrate their potential biological effects, purified standards were purchased, and their antagonistic activity against *Verticillium dahliae* was investigated. Example 2: Inhibitory effect of volatile organic compounds from purple-blue-producing bacteria on Verticillium dahliae 1. Inhibitory effect on the growth of Verticillium dahliae colonies To determine the effect of volatile compounds on *Verticillium dahliae*, 20 mL of PDA medium was placed in a 70 mL petri 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 petri 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 concentrations ranging from 1.5625 to 3200 μL / L. The culture conditions and inhibition rate calculations were the same as before. After 15 days of incubation on PDA medium, different concentrations of the standard products showed inhibitory effects on *Verticillium dahliae* hyphae. 1-Octen-3-one exhibited the highest activity, followed by 2-octen-1-ol and 1-octaen-3-ol, while 3-octanone showed the lowest activity. The EC50 values ​​were 14.59, 240.1, 432.9, and 3923 μL / L, respectively. The inhibitory activity of volatile organic compounds with different structures may be due to the greater activity of the ketone group (11-ol) than the alcohol group, as well as differences in the positions of the carbon-carbon double bond (13-ol) and the alcohol group (E and 11-ol). The inhibitory effect of 1-octen-3-ol was stronger than that of 3-octanone, consistent with the inhibitory results of four other pathogenic fungi (*Rhizoctonia solani*, *Fusarium oxysporum*, *Phytophthora infestans*, and *Phytophthora brown rot*). Adding 1 μL of volatile organic compounds inhibited the germination of Verticillium dahliae spores. However, the inhibitory effect of 3-octanone was relatively low compared to other volatile organic compounds. 2. Inhibitory effect on the germination of Verticillium dahliae conidia A 200 μL culture system was established by adding 10 μL of methanol solution containing 1 μL of volatile organic compounds, 90 μL of LPDB solution, and 100 μL of spore suspension (1 x 10⁻⁶ spores) to a 1.8 mL EP tube. The system was then incubated at 150 rpm and 25 °C. The inhibition rate was calculated as follows: Inhibition rate (%) = (Spore germination rate of control group - Spore germination rate of treatment group) / (Spore germination rate of control group) x 100. The germination rates of the control group and treatment group were calculated at 24 h and 48 h in the same field of view. The main volatile organic compound (VOC) component of CEF642N, 1-octen-3-one, reduced mycelial development in *Verticillium dahliae*. One treatment with 800 μL / L 1-octen-3-one was administered. The inhibition rate gradually decreased with increasing treatment time. This indicates that 1-octen-3-one may also induce some responses in *Verticillium dahliae*, thereby reducing the inhibitory effect of external stress. In the cross-conversion test between CEF642N and Vd076, the total VOCs produced by CEF642N gradually increased with increasing treatment time, and the inhibition rate of *Verticillium dahliae* also gradually increased. Scanning electron microscopy images showed that the control group mainly consisted of mycelia (Fig. 1A), while the 1-octen-3-one-treated group contained some mycelia and spores, with some spores atrophied (Fig. 1B). The VOCs-treated group consisted mainly of spores, which also atrophied (Fig. 1C), indicating that VOCs inhibited spore germination. The total volatile organic compounds (TVOC) and 1-octen-3-one in CEF642N affect the germination and development of Verticillium dahliae spores and the growth of mycelium. Transmission electron microscopy revealed that, compared to the control group, 1-octen-3 one and total volatile organic compounds (VOCs) in CEF642N caused severe organelle damage, mitochondrial degeneration, and cavitation. In our study, VOCs reduced the growth of *Verticillium dahliae* hyphae, spore germination, and the internal structure of pathogen cells (Figure 2). Example 3: Reparasitism of *Verticillium dahliae* CEF642N on *Verticillium dahliae* Vd076 The timeline of fungal parasitism was consistent with the description of the confrontation culture of CEF642N and Vd076 above. CEF642N and Vd076 (5 mm in diameter) were inoculated into PDA medium. Additionally, PDA medium coated with Vd076 spores was cultured on three different media for 7 days. The diameter of CEF642N colonies was measured, and photographs were taken every 3 days. The confrontation culture of CEF642N and Vd076 was performed, and the parasitic fungal portion in the confrontation culture was examined using scanning electron microscopy. On day 12 of the confrontation culture, CEF642N and Vd076 were brought into contact. With prolonged co-culture, the coverage area of ​​CEF642N on Vd076 steadily increased, indicating an increase in fungal parasitism (Figure 3). During the co-culture of CEF642N and Vd076, CEF642N may produce antibiotics to kill the pathogen and secrete cell wall-degrading enzymes, including chitinase and β-1,3-glucanase, leading to fungal parasitism. When CEF642N was inoculated into a medium already inoculated with *Verticillium dahliae*, it was observed that CEF642N could grow on Vd076 hyphae and on media covered with Vd076 spores. This indicates that CEF642N can bioparasitize on Vd076, utilizing the nutrients of *Verticillium dahliae* for its own development. 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 utilize multiple mechanisms to collect nutrients from a living host. Scanning electron microscopy images of CEF642N(Tp) cultured alone on PDA medium showed numerous protrusions on its hyphae (Fig. 4A), while the hyphae of Vd076(Vd) were smooth (Fig. 4B). In the parasitic portion of the confront culture, the hyphae of CEF642N could grow parallel to Verticillium dahliae (Fig. 4C, D). In summary, this invention screened the inhibitory effect of the purplish-blue bacterium CEF642N on *Verticillium dahliae*. It inhibits the growth of *Verticillium dahliae* through volatile organic compounds and non-volatile secondary metabolites, and also exhibits fungal parasitism against *Verticillium dahliae*. Genomic sequencing analysis revealed that CEF642N contains a large number of lipid metabolism genes, which may be precursors for antagonizing volatile organic compounds, as well as genes related to secondary metabolites such as terpenes and polyketides. Comparative genomic analysis showed that CEF642N is evolutionarily closer to *Bambusa stolonifera*, with a more similar chemical phenotype. Carbohydrate-active enzymes, after gene deletion and new functionalization, may possess greater cell wall degradation functions. The mechanism of action of CEF642N against *Verticillium dahliae* involves early volatile organic compounds targeting the mitochondria and cell membrane of the pathogen, followed by the action of non-volatile secondary metabolites, ultimately leading to parasitism upon contact. The results indicate that CEF642N possesses multiple antagonistic effects and has broad application prospects in the control of cotton verticillium wilt.

Claims

1. A strain of Talaromyces purpureogenus, characterized in that, The preservation number of the Janthinaceae bacterium is CCTCC NO: M20242093.

2. Use of the Janthinaceae bacterium in claim 1 in the preparation of a biological control agent for preventing and treating Verticillium dahliae-induced cotton Verticillium wilt.

3. Use according to claim 2, wherein the compound is ###0002### The biological control agent is a liquid preparation.

4. A method of controlling cotton Verticillium wilt, comprising applying to the locus of the cotton plant a composition comprising a fungicidally effective amount of a compound of Formula (I) or a salt thereof. The method is to use the Janthinaceae bacterium in claim 1 to prevent and treat Verticillium dahliae-induced cotton Verticillium wilt.

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