Talaromyces sp. and its use in antagonizing fusarium

Abstract

The application discloses a Talaromyces sp. and application thereof in antagonizing Fusarium. The Talaromyces sp. QHSH-14B is preserved in the China General Microbiological Culture Collection Center, and has a preservation number of CGMCC NO.40625. The Talaromyces sp. QHSH-14B and sterile fermentation liquor thereof have antagonistic inhibition effects on Fusarium. It is known through confrontation culture of the Talaromyces sp. QHSH-14B and sterile fermentation liquor thereof and Fusarium xylariophilum that the inhibition rate of the Talaromyces sp. QHSH-14B on the Fusarium xylariophilum is 66.14%, and the inhibition effect is strong; the inhibition rate of the sterile fermentation liquor of the Talaromyces sp. QHSH-14B on the Fusarium xylariophilum is 20.77% to 29.60%, and the inhibition effect is better. The Talaromyces sp. QHSH-14B has an influence on mycelium growth and morphology of the Fusarium xylariophilum, and has a good inhibition and biocontrol effect on the Fusarium xylariophilum.

Description

Technical Field

[0001] This invention belongs to the field of microbial technology and relates to a strain of *Basilella* and its application in antagonistic *Fusarium*. Background Technology

[0002] Fusarium sp. is a soil-borne fungal disease that is widespread in nature. It is one of the top ten fungal diseases among plant diseases and can infect nearly a hundred plant species. It is known as "plant cancer". In the early stages of infection, it can cause symptoms such as wilting, leaf withering and inability to unfold, and root and stem rot. In severe cases, it can lead to the death of the entire plant, which seriously restricts agricultural development and causes a lot of economic losses. Pathogenic Fusarium fungi are numerous and diverse, including Fusarium solani, Fusarium oxysporium, Fusarium concolor, Fusarium moniliforme, Fusarium sambucinum, Fusarium graminearum, and Fusarium equiseti. Among them, Fusarium sambucinum can cause root rot in potatoes, astragalus, alfalfa, and stem rot in Cistanche deserticola, and is a common pathogenic fungus.

[0003] Existing technologies for controlling soil-borne diseases caused by Fusarium include chemical control, plant-derived fungicides, and biological control. Chemical control methods include soil disinfection using soil disinfectants such as cymoxanil and calcium cyanamide during greenhouse fallow periods; or root irrigation with fungicides such as metalaxyl, fludioxonil, copper oxychloride, prochloraz, methyl thiophanate, and pentachloronitrobenzene for infected plants. However, long-term use of chemical agents can easily lead to drug resistance in pathogens and also cause environmental pollution. Patent CN106719645A discloses a plant-derived fungicide for controlling potato dry rot and its preparation method. This plant-derived fungicide is a solution of ABBA extracted from Dryopteris crassirhizoma, with a concentration of 0.1–2.0 mg / mL. This plant-derived fungicide has an inhibitory effect on the main pathogens of potato dry rot, including Fusarium oxysporum, Fusarium oxysporum oatum, Fusarium solani, and Fusarium solani blue variant.

[0004] In recent years, the use of biological control methods to prevent and control plant diseases has gradually become a research hotspot. Its advantages of low cost and being environmentally friendly and residue-free align with the public's pursuit of a healthy life. Currently, the main types of biocontrol agents used to control plant diseases include bacteria, fungi, and actinomycetes. Fungi are widely distributed in soil and on plant surfaces, easily isolated from plant debris, seeds, and bulbs, and have antagonistic effects against various pathogenic fungi and bacteria, thus they have been used for the control of plant diseases. Summary of the Invention

[0005] To further promote research on biocontrol fungi for controlling Fusarium, this invention provides a basket-shaped fungus that can effectively inhibit the growth of Fusarium fungi, specifically Fusarium elderberry, thus providing microbial resources for controlling plant diseases caused by Fusarium.

[0006] To achieve the technical objective of this invention, in one aspect, this invention provides a strain of *Talaromyces* sp. QHSH-14B. *Talaromyces* sp. QHSH-14B was deposited on May 15, 2023, at the China General Microbiological Culture Collection Center (CGMCC), with accession number CGMCC NO. 40625. The ITS1 product sequence of *Talaromyces* sp. QHSH-14B is shown in SEQ ID NO. 1; the ITS4 product sequence of *Talaromyces* sp. QHSH-14B is shown in SEQ ID NO. 2.

[0007] Furthermore, the strain QHSH-14B of this invention belongs to the genus *Basilaria*.

[0008] Specifically, this invention identifies strain QHSH-14B as belonging to the genus *Basilaria* by examining its morphology, homology, and phylogenetic tree.

[0009] Furthermore, the growth curve of *Talaromyces* sp. QHSH-14B of this invention shows that growth is slow from 0 to 1 day after inoculation, rapid growth begins after 1 day, the logarithmic growth phase is from 1 to 6 days, and the total mycelial weight begins to decrease after 6 days, entering the growth decline phase. The culture conditions for *Talaromyces* sp. QHSH-14B are: modified Martin liquid medium, 28°C, shaking at 180 rpm.

[0010] Furthermore, the present invention's *Talaromyces* sp. QHSH-14B and its sterile fermentation broth have the effect of inhibiting *Fusarium* species *Eucomyces*.

[0011] Specifically, this invention utilizes confrontation culture of *Talaromyces sp. QHSH-14B* and *Fusarium oxysporum*, confrontation culture of *Talaromyces sp. QHSH-14B* sterile fermentation broth and *Fusarium oxysporum*, and observation of the morphology of *Fusarium oxysporum* after confrontation culture. These findings demonstrate that *Talaromyces sp. QHSH-14B* has an inhibitory effect on *Fusarium oxysporum*. The confrontation culture of *Talaromyces sp. QHSH-14B* with *Fusarium oxysporum* showed an inhibition rate of 66.14%, indicating a strong inhibitory effect. After confrontation culture of aseptic fermentation broth of *Talaromyces sp. QHSH-14B* with *Fusarium oxysporum*, it was found that the aseptic fermentation broth of *Talaromyces sp. QHSH-14B* inhibited *Fusarium oxysporum* by 20.77%–29.60%, showing a good inhibitory effect. Observation of the morphology of *Fusarium oxysporum* after confrontation culture showed that *Talaromyces sp. QHSH-14B* affected the mycelial growth and morphology of *Fusarium oxysporum*, effectively inhibiting its growth and demonstrating a good antibacterial and control effect.

[0012] On the other hand, the present invention seeks protection for the use of *Talaromyces sp. QHSH-14B* in the prevention and control of plant diseases caused by *Fusarium*.

[0013] Compared with the prior art, the technical solution provided by the present invention has at least the following beneficial effects or advantages:

[0014] The *Talaromyces* sp. QHSH-14B isolated and purified in this invention exhibits good inhibitory effects on the growth of *Fusarium*. *Talaromyces* sp. QHSH-14B showed a 66.14% inhibition rate against *Fusarium sibiricum*, demonstrating a strong inhibitory effect. The aseptic fermentation broth of *Talaromyces* sp. QHSH-14B showed an inhibition rate of 20.77%–29.60% against *Fusarium sibiricum*, indicating good inhibitory activity. *Talaromyces* sp. QHSH-14B can induce varying degrees of shrinkage, shriveling, breakage, and rough, uneven cell formation in *Fusarium sibiricum* hyphae; in severe cases, a large amount of material may deposit on the hyphae. Some hyphae show tight adhesion, and the spore morphology changes make it difficult to observe the typical sickle-shaped pattern. This effectively inhibits the growth of *Fusarium sibiricum* and provides a good antibacterial and control effect against *Fusarium sibiricum*, offering a microbial resource for the biological control of plant diseases caused by *Fusarium*. Attached Figure Description

[0015] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention.

[0016] Figure 1 This is a colony diagram of *Talaromyces* sp. QHSH-14B.

[0017] Figure 2 Phylogenetic tree of Talamoyces sp. QHSH-14B.

[0018] Figure 3 This is a growth curve of Talamomyces sp. QHSH-14B.

[0019] Figure 4 This image shows the growth inhibition effect of *Talaromyces sp. QHSH-14B* on *Fusarium oxysporum*. A is the blank control; B is a confrontation culture of *Talaromyces sp. QHSH-14B* and *Fusarium oxysporum*.

[0020] Figure 5 The graph shows the growth inhibition effect of different concentrations of *Talaromyces sp.* QHSH-14B on *Fusarium oxysporum*. A is the control group; B is the 5% concentration group; C is the 10% concentration group; D is the 15% concentration group; and E is the 20% concentration group.

[0021] Figure 6 This is a diagram of the hyphal morphology of the control group, *Fusarium oxysporum*. a shows conidia attached to the hyphae at the spore tip or spore body.

[0022] Figure 7 This is a diagram of the hyphal morphology of the control group, *Fusarium oxysporum*. (a) shows conidia with pointed ends and a slightly curved, sickle-shaped spore body.

[0023] Figure 8 This image shows the morphological changes of *Fusarium septum* hyphae during a confrontation culture with *Talaromyces* sp. QHSH-14B. b shows hyphae with varying degrees of shrinkage; c shows hyphae with varying degrees of shriveling; d shows hyphae with varying degrees of breakage.

[0024] Figure 9This image shows the morphological changes of *Fusarium spp.* hyphae during a confrontation culture between *Fusarium spp.* sp. QHSH-14B. e shows rough, uneven mycelial structures with significant material deposition on the hyphae; f shows tightly packed hyphae; g shows spores where the typical sickle shape is difficult to observe. Detailed Implementation

[0025] The technical solution of the present invention will be described below with reference to embodiments. However, the present invention is not limited to the following embodiments. Unless otherwise specified, the experimental methods and detection methods described in each embodiment are conventional methods; unless otherwise specified, the reagents and materials can be purchased commercially.

[0026] The modified Martin liquid culture medium was purchased from Qingdao High-tech Park Haibo Biotechnology Co., Ltd., product number HB5222.

[0027] Modified Martin solid medium: Based on modified Martin liquid medium (5.0g peptone, 1.0g dipotassium hydrogen phosphate, 0.5g magnesium sulfate, 2.0g yeast extract and 20.0g glucose), add 20g agar (Beijing Boao Tuoda Technology Co., Ltd.), bring the volume to 1L with distilled water, and sterilize at 121℃ for 15min.

[0028] Example 1

[0029] This embodiment provides a method for the isolation, purification, and identification of Talaromyces sp. QHSH-14B, specifically including the following steps:

[0030] 1. Separation and purification

[0031] Fresh wild Sanghuang (collected in October 2019 from Qinghai Province) was used for surface disinfection: rinsing thoroughly with clean water, cutting off disease-free parts, rinsing 4 times with clean water, rinsing with 75% alcohol for 5 minutes, soaking in 5% sodium hypochlorite for 10 seconds, and rinsing 10 times with sterile water. Surface moisture was absorbed with sterile filter paper, the surface was cut off with a sterile blade, and the interior was cut into small pieces and transplanted into modified Martin's medium (containing 150 mg / L penicillin) for incubation at 28°C. Purification was performed using the streak plate method to obtain multiple single purified microbial strains QHSH-14B.

[0032] 2. Morphological identification

[0033] Strain QHSH-14B was inoculated onto modified Martin medium and cultured at 28°C for 5–7 days. Single colony morphology was then observed. Figure 1 The colonies are initially white, then turn dark green or blackish-green, with a fluffy appearance and obvious wrinkles. As the culture time increases, the colony color deepens, the culture medium does not change color, and the bacteria are easy to scrape off.

[0034] 3. Molecular identification

[0035] 1) Inoculate strain QHSH-14B into modified Martin liquid medium and culture on a shaker at 28℃ and 180r / min for 5 days.

[0036] 2) After centrifugation, take a small amount of bacterial cells and extract the genomic DNA of strain QHSH-14B using a fungal genomic DNA extraction kit (purchased from Wuxi Biotech Co., Ltd., catalog number: B004009020). The extraction steps are as per the instructions of the fungal genomic DNA extraction kit.

[0037] 3) Using the genomic DNA of strain QHSH-14B as a template, PCR amplification was performed using universal primers ITS1 and ITS4. The primer sequences are as follows:

[0038] ITS1(5'-TCCGTAGGTGAACCTGCGG-3');

[0039] ITS4(5'-TCCTCCGCTTATTGATATGC-3').

[0040] The PCR amplification reaction system (50 μL) includes: 10 μL of 5× buffer; 4 μL of dNTP; 1 μL of upstream primer ITS1 (10 μM); 1 μL of downstream primer ITS4 (10 μM); 0.5 μL of Prime STAR; 1 μL of template (1 μM); and 32.5 μL of ddH2O.

[0041] The PCR reaction conditions were: 95℃ pre-denaturation for 5 min; 94℃ denaturation for 30 s, 55℃ annealing for 30 s, 72℃ extension for 2 min, 30 cycles; 72℃ extension for 5 min.

[0042] 4) The amplification products (i.e., the ITS of strain QHSH-14B) were sequenced (Beijing Youji Technology Co., Ltd.), and the ITS1 product sequence of strain QHSH-14B is shown in SEQ ID NO.1; the ITS4 product sequence of strain QHSH-14B is shown in SEQ ID NO.2.

[0043] 5) The obtained sequences were compared with the NCBI nucleic acid database using BLAST for homology analysis. Some highly similar strains were selected and subjected to phylogenetic analysis with strain QHSH-14B. A phylogenetic tree was constructed using the neighbor-joining method in MEGA 7.0 software. Figure 4 ).

[0044] Based on morphological characteristics, homology, and phylogenetic tree, QHSH-14B was identified as belonging to the genus *Basilaria*.

[0045] Example 2

[0046] This embodiment provides a method for determining the growth curve of Talaromyces sp. QHSH-14B, specifically including the following steps:

[0047] Single colonies of *Talaromyces* sp. QHSH-14B were inoculated into Erlenmeyer flasks containing 150 mL of modified Martin broth and activated by shaking at 28 °C and 180 rpm. A 2% (v / v) inoculum was then inoculated into 250 mL Erlenmeyer flasks containing 150 mL of modified Martin broth and incubated at 28 °C and 180 rpm. Samples were taken every 24 h, mycelia were collected by filtration, dried in an oven at 45 °C, and weighed. Each treatment was performed in triplicate. The growth curve of *Talaromyces* sp. QHSH-14B was determined by the dry weight method. Figure 2 ).

[0048] Depend on Figure 2 It can be seen that Talamomyces sp. QHSH-14B grows slowly from 0 to 1 day after inoculation, with a short growth lag phase. After 1 day, it begins to grow rapidly and enters the logarithmic growth phase. Days 1 to 6 are the logarithmic growth phase of Talamomyces sp. QHSH-14B. After 6 days, the total weight of mycelium begins to decrease and enters the growth decline phase. No obvious growth stationary phase was observed.

[0049] Example 3

[0050] This embodiment provides a confrontation culture between *Talaromyces* sp. QHSH-14B and *Fusarium oxysporum*, specifically including the following steps:

[0051] Using *Fusarium solani* as an indicator bacterium, the three-point confrontation culture method was employed to screen for *Talaromycess p. QHSH-14B*, which showed good inhibitory effect on the growth of *Fusarium solani*. Activated *Fusarium solani* mycelial blocks were collected using the blunt end of a 1 mL pipette tip as a sterile punch and inoculated into the center of a 9 cm diameter modified Martin solid medium. *Talaromycess p. QHSH-14B* was inoculated with bamboo sticks at three points: above, left, and right, and 2–3 cm from the center of the plate. A blank control was prepared by inoculating only the pathogen without any biological control. The plates were incubated at 28°C for 5–7 days. When the hyphal diameter of the control plate reached three-quarters of the plate diameter, the hyphal diameter (cm) was measured using the cross-cross method, and the inhibition rate of *Talaromycess p. QHSH-14B* on *Fusarium solani* was calculated. Each treatment was performed in triplicate.

[0052] Inhibition rate (%) = (Coronary diameter of control group - Colony diameter of experimental group) / Colony diameter of control group × 100%

[0053] The *Fusarium solani* strain was provided by the Laboratory of Microbiology and Biochemical Pharmacy, Ningxia Medical University.

[0054] The inhibitory effect of Talaromyces sp. QHSH-14B on Fusarium oxysporum is as follows: Figure 4 As shown, the colony diameter in the control group was 6.94±0.29 cm, and the colony diameter in the experimental group was 2.35±0.08 cm. Calculations showed that Talaromycess p. QHSH-14B inhibited Fusarium tumefaciens by 66.14%, demonstrating a highly effective inhibitory effect on the mycelial growth of Fusarium tumefaciens.

[0055] Example 4

[0056] This embodiment demonstrates the inhibitory effect of aseptic fermentation broth of *Talaromyces* sp. QHSH-14B on the mycelial growth of *Fusarium oxysporum*, specifically including the following steps:

[0057] Talaromyces sp. QHSH-14B was inoculated into modified Martin liquid medium and activated by shaking culture at 28°C and 180 rpm for 4 days. The inoculum was then inoculated into a 250 mL Erlenmeyer flask containing 150 mL of Martin liquid medium at a 2% (v / v) inoculation rate and shaken for 4 days to obtain the fermentation broth. The supernatant was collected by filtration and filtered through a 0.22 μm sterile filter membrane to obtain the sterile fermentation broth.

[0058] After autoclaving the modified Martin solid medium and cooling it to 50–60°C, 5%, 10%, 15%, and 20% (v / v) of sterile fermentation broth of *Talaromyces* sp. QHSH-14B were added respectively. After thorough mixing, the mixture was poured into 9 cm plates. 30 mL of Martin solid medium without sterile fermentation broth was used as a control. After the mixed medium solidified, *Fusarium oxysporum* was inoculated into the center of the plate by punching holes. The plates were incubated at 28°C for 5–7 days. When the mycelial diameter of the control plate reached three-quarters of the plate diameter, the mycelial diameter (cm) was measured using the cross-cross method. Three replicates were performed for each treatment, and the inhibition rate of different concentrations of sterile fermentation broth on the mycelial growth of *Fusarium oxysporum* was calculated (Table 1).

[0059] The aseptic fermentation broth of *Talaromyces* sp. QHSH-14B showed the following inhibitory effect on the mycelial growth of *Fusarium oxysporum*: Figure 5 As shown.

[0060] Table 1: Inhibitory effect of aseptic fermentation broth of *Talaromyces* sp. QHSH-14B on *Fusarium oxysporum*

[0061] Colony diameter (cm) 6.79±0.10 5.06±0.19 5.15±0.02 5.38±0.08 4.78±0.10 Inhibition rate (%) / 25.48％ 24.15％ 20.77％ 29.60％

[0062] Table 1 shows that the aseptic fermentation broth of *Talaromyces* sp. QHSH-14B has an inhibitory effect on *Fusarium oxysporum*, with an inhibition rate of 20.77%–29.60%. It is worth noting that the colony diameter is measured using the longest side. Figure 5 It can be seen that the growth regularity of *Fusarium spp.* in the fermentation broth of 10% and 15% *Talaromyces* sp. QHSH-14B is lacking, so the values ​​are relatively large. However, it can be seen that the antibacterial effect is concentration-dependent. As the concentration of QHSH-14B increases, the colony density and mycelial state of *Fusarium spp.* become worse.

[0063] Example 5

[0064] This embodiment observes the morphological changes of *Fusarium graminearum* after confrontation culture with *Talaromyces sp. QHSH-14B*, specifically including the following steps:

[0065] 1) Talaromyces sp. QHSH-14B and Fusarium elderberry were cultured in confrontation for 7 days, using the same method as in Example 3. The pathogenic bacteria block near the inhibition zone was cut with a sterile scalpel, and the size of the bacterial block was 0.5cm square.

[0066] 2) Place the bacterial block into a sterile 12-well plate, add 2% glutaraldehyde solution, and fix overnight at 4°C.

[0067] 3) Add 0.1M phosphate buffer and wash three times, 10 minutes each time.

[0068] 4) Add osmium tetroxide fixative and fix in a 4°C refrigerator for 2 hours.

[0069] 5) Add 0.1M phosphate buffer and wash three times, 10 minutes each time.

[0070] 6) Dehydrate with 30%, 50%, 70%, 90% and 100% ethanol respectively, for 10 minutes each time.

[0071] 7) Replace the bacterial block three times with 70% tert-butanol solution for 10 minutes each time, and then replace it twice with 100% tert-butanol solution. The first replacement is for 10 minutes, and the second replacement is with a small amount of tert-butanol to cover the bacterial block. After the replacement is completed, put the sample in a -20℃ refrigerator for pre-cooling for 20 minutes.

[0072] 8) After drying in a vacuum desiccator and sputtering, place the prepared sample under a Hitachi S-3400N scanning electron microscope and observe the hyphal morphology at 10 kV and appropriate magnification. Figures 6-9 ).

[0073] The *Fusarium solani* strain that was not inoculated with *Talaromyces* sp. QHSH-14B was used as a control, and the other treatments were the same.

[0074] Depend on Figure 6 It can be seen that the hyphae in the control group are smooth, plump, and uniform in size, and the conidia are attached to the hyphae by the spore tips or spore bodies. Figure 6 a) The spores are pointed at both ends, and the spore body is slightly curved and sickle-shaped. Figure 7 a). The hyphae treated with *Talaromyces* sp. QHSH-14 showed varying degrees of shrinkage. Figure 8 b) Shriveled Figure 8 c) Fracture Figure 8 d), the fungal cells are rough and uneven; in severe cases, a large amount of material may even be deposited on the hyphae. Figure 9 e), some hyphae also show close adhesion ( Figure 9 f) It is difficult to observe typical sickle-shaped spore morphological changes. Figure 9 The results (g) indicate that Talamyces p. QHSH-14B affects the mycelial growth and morphology of Fusarium tumefaciens, effectively inhibiting its growth and demonstrating good antibacterial and preventative effects.

[0075] As described above, the basic principles, main features, and advantages of the present invention have been well described. The above embodiments and specifications are merely descriptions of preferred embodiments of the present invention, and the present invention is not limited to the above embodiments. Various changes and improvements made to the technical solutions of the present invention by those skilled in the art without departing from the spirit and scope of the present invention should fall within the protection scope defined by the present invention.

Claims

1. A strain of basket-shaped fungus ( Talaromyces sp. QHSH-14B, characterized in that, It was deposited on May 15, 2023, at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC NO.40625.

2. The basket-shaped bacteria QHSH-14B according to claim 1, characterized in that, The basket-shaped bacterium QHSH-14B has an inhibitory effect on Fusarium oxysporum.

3. The basket-shaped bacteria QHSH-14B according to claim 1, characterized in that, The culture conditions for the basket-shaped bacteria QHSH-14B are: cultured in modified Martin solid medium at 28°C for 5-7 days; The modified Martin solid medium comprises: 5.0 g / L peptone, 1.0 g / L dipotassium hydrogen phosphate, 0.5 g / L magnesium sulfate, 2.0 g / L yeast extract, 20.0 g / L glucose, and 20 g / L agar.

4. The use of the basket fungus QHSH-14B according to any one of claims 1 to 3 in the prevention and control of plant diseases caused by Fusarium oxysporum.

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