Penicillium brevicompactum and application thereof in biological control and growth promotion

Abstract

The invention belongs to the field of microorganisms, and particularly provides a Penicillium brevicompactum Q-9 strain (Penicillium brevicompactum Q-9), which is derived from garlic rhizosphere soil, is identified as non-pathogenic Penicillium brevicompactum, is preserved in China General Microbiological Culture Collection Center (CGMCC) on April 11, 2024, and has a preservation number of CGMCC No.41153. The Penicillium brevicompactum Q-9 strain is applied to the field of microorganisms. The Q-9 strain grows rapidly, can secrete phosphate-solubilizing enzyme, has broad-spectrum antagonistic activity, and has a remarkable inhibition effect on various plant pathogenic fungi (such as fusarium oxysporum and cylindrosporium), and the highest bacteriostasis rate reaches 77.32%. The strain can significantly promote plant growth and improve crop biomass. The invention also provides application of the strain in the aspects of plant disease control and plant growth promotion.

Description

Technical Field

[0001] The present invention relates to the field of microorganisms, and in particular to a non-pathogenic Penicillium brevicaulis and its application in pest control and growth promotion. Background Art

[0002] The Penicillium brevi-compactum Q-9 strain is recruited from the sulfur-containing compounds of garlic. It has growth-promoting properties and can effectively antagonize some pathogens of serious diseases of Solanaceae and Panax notoginseng. It can also cooperate with the broad-spectrum biocontrol fungus Pseudomonas aeruginosa.

[0003] Tomatoes, a member of the Solanaceae family, are a key vegetable crop that has rapidly grown in my country, making the country one of the leading growers and producers. Rich in nutrients such as vitamins and lycopene, which can replenish essential human functions, they are highly sought after. Besides being affected by natural conditions such as geography and weather, tomato cultivation is also subject to the inevitable threat of disease, which varies widely and can, in severe cases, cause widespread yield losses. Tomato early blight and wilt are both significant global diseases of tomatoes, with high incidence rates and significant yield losses. Tomato wilt, caused by Fusarium oxysporum, is characterized by significant damage and difficulty in prevention and control. Current chemical control methods can cause environmental pollution and pesticide residues, and long-term use of chemical agents can also lead to antibiotic resistance and food safety issues.

[0004] Panax notoginseng, a perennial plant of the genus Panax in the Araliaceae family, is cultivated in shade under no-tillage conditions. It prefers warm, humid weather and is a renowned Chinese medicinal herb. Considered a rare and endemic Chinese herb, it is revered as "golden beyond compare" and "southern divine grass" for its exceptional medicinal value. Limited by its variety and unique growing environment, international research reports on this herb are limited. However, it boasts benefits such as promoting blood circulation, dispersing blood stasis, relieving swelling and pain, and providing antioxidant and anti-aging benefits. Yunnan, the primary production area of Panax notoginseng, has seen significant deterioration in the rhizosphere soil ecosystem due to years of large-scale monoculture. Continuous cropping disorder is a serious problem, severely impacting local economic development. Continuous cropping disorder refers to the imbalance of soil microbial communities caused by the continuous planting of the same crop species or family on the same land, even under normal management conditions. This can lead to reduced seedling emergence, increased pests and diseases, and ultimately compromised product quality. Root rot is one of the most serious diseases affecting Panax notoginseng, accounting for over 70% of all its diseases. It can reduce Panax notoginseng yield by 5% to 70%, or even completely eliminate the crop. Furthermore, it often takes more than 10 years before the soil can be replanted. These issues severely impact the sustainable cultivation and yield of Panax notoginseng, necessitating the urgent need for effective measures to address this challenge.

[0005] Microbial synergy refers to the phenomenon in which two or more microorganisms interact in a specific environment, thereby jointly influencing their growth, metabolism, or biological functions. This synergistic relationship is widely present in nature, such as in soil, oceans, plants and animals, and the human intestinal microbiome. Two microbial strains form complex ecological networks through nutrient exchange, metabolite secretion, and signal transmission, which have a significant impact on environmental adaptation, resource utilization, and the expression of specific functions. The study of microbial synergy not only reveals the ecological relationships between microorganisms but also provides an important application foundation for agriculture, medicine, and environmental science.

[0006] Microorganisms can secrete plant hormones, which can promote antibiotic resistance, parasitize plants, and enhance plant resistance. However, most current biocontrol agents offer strong antagonistic effects but poor broad-spectrum control, while those with strong broad-spectrum control have poor antagonistic effects. Therefore, developing biocontrol agents that combine resistance and broad-spectrum protection presents another challenge in biological control. Summary of the Invention

[0007] In order to solve the above technical problems, the present invention provides the following technical solutions:

[0008] First, the first aspect of the present invention provides a strain of Penicillium brevicompactum Q-9. The strain has been identified as Penicillium brevicompactum, is non-pathogenic, and has been deposited in the General Microbiology Center of the China Microorganism Culture Collection Administration (CGMCC) with a deposit number of CGMCC No. 41153 (deposit date: April 11, 2024). The strain grows rapidly on potato dextrose agar (PDA), and the colonies are blue-green velvety with white edges. It can secrete active substances such as phospholytic enzymes.

[0009] A second aspect of the present invention provides the use of the aforementioned Penicillium breviscompactum Q-9 strain for controlling plant diseases. This strain exhibits antagonistic activity against a variety of plant pathogenic fungi and can be used to control a variety of crop diseases, including tomato wilt, tomato early blight, and Panax notoginseng root rot. By formulating the Q-9 strain into a biocontrol agent and applying it to plants or soil, it effectively inhibits pathogen growth and reduces the occurrence of diseases.

[0010] A third aspect of the present invention provides the use of the aforementioned Penicillium breviscompactum Q-9 strain for promoting plant growth. This strain significantly promotes plant growth and development by secreting plant growth stimulating substances and increasing soil nutrient availability (such as dissolving insoluble phosphorus). For example, pepper plants treated with the Q-9 strain exhibited significantly higher plant height and biomass than untreated controls.

[0011] A fourth aspect of the present invention provides a biological preparation containing the Q-9 strain of Penicillium breviscompactum and a method for preparing the same. The biological preparation contains the Q-9 strain as the active ingredient and can be formulated into dosage forms such as suspensions and wettable powders. The preparation method comprises culturing and amplifying the Q-9 strain to obtain a large number of cells or spores, and, if necessary, mixing the fermentation liquid or cells with a carrier to prepare a product. The biological preparation can be used for soil treatment, seed coating, or plant spraying to prevent and control diseases and promote crop growth.

[0012] A fifth aspect of the present invention provides a technical solution for the combined control of plant diseases using the Q-9 strain of Penicillium breviscompactum and another broad-spectrum biocontrol bacterium, Pseudomonas aeruginosa. By combining the Q-9 strain with Pseudomonas aeruginosa YNAU-A5, the control spectrum can be further expanded and the efficacy enhanced. The two strains exhibit no antagonistic interactions when co-cultured, exhibiting symbiotic compatibility and synergistic inhibitory effects against target pathogens, thus providing a new combined strategy for the biological control of crop diseases.

[0013] Compared with the prior art, the present invention has significant beneficial effects:

[0014] 1. Broad-spectrum and highly effective disease control: Penicillium breviscompactum Q-9 exhibits strong antagonistic activity against a wide range of plant pathogens. Experimental results showed that the inhibition rate against 17 common plant pathogens tested ranged from 39.24% to 77.32%, with the highest inhibition rate reaching 77.32% against Rhizoctonia solani, the pathogen of rice sheath blight. Therefore, this strain can be used to control a wide range of crop fungal diseases, overcoming the limited control spectrum of single biocontrol agents.

[0015] 2. Significant Plant Growth-Promoting Effect: Penicillium breviscompactum Q-9 can secrete beneficial enzymes such as phosphatases, promoting the release of insoluble phosphorus from the soil and improving nutrient absorption and utilization by plants. Potted plant trials showed that pepper plants treated with Q-9 grew significantly taller and had significantly higher biomass than the untreated control group, demonstrating its significant growth-promoting effect. Therefore, the application of Q-9 can simultaneously promote crop growth and increase yields while preventing disease.

[0016] 3. Good safety and compatibility: The Q-9 strain is derived from plant rhizosphere soil and is inherently non-pathogenic, safe, and environmentally friendly, harmless to crops and the environment, making it suitable for use as a biopesticide or biofertilizer. Furthermore, the Q-9 strain can synergize with other beneficial microorganisms (such as Pseudomonas YNAU-A5) without antagonism. This cross-kingdom combination of fungi and bacteria offers new possibilities for biocontrol, leveraging the strengths of multiple microorganisms to enhance the stability and broadness of control effects.

[0017] In summary, the Penicillium brevicaulis Q-9 strain provided by the present invention has both broad-spectrum antibacterial and growth-promoting effects, can be used to develop highly effective biological control agents, reduce dependence on chemical pesticides, and has important agricultural application value and promotion prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] The accompanying drawings are used to provide a further understanding of the present invention and constitute a part of the specification. Together with the embodiments of the present invention, they are used to explain the present invention and do not constitute a limitation of the present invention. In the accompanying drawings:

[0019] Figure 1 The morphological characteristics of Penicillium breviscompactum Q-9 strain on PDA medium, where a and b are colony morphologies, and c is the morphology of hyphae and conidia;

[0020] Figure 2 The transparent aperture for the production of phosphalytic enzyme by Penicillium breviscompactum Q-9 strain;

[0021] Figure 3 The plates were cultured with three pathogens of Penicillium breviscompactum Q-9 for 7 days. The top three pictures are the control group, and the bottom three pictures are the plates with Penicillium breviscompactum Q-9 added respectively.

[0022] Figure 4 A: Colony image of Q-9 on PDA; Figure 4 B: Q-9 can cooperate with Pseudomonas; Figure 4 C: Pseudomonas antagonistic to the pathogen of Panax notoginseng rust, Cylindrocarpon destructans;

[0023] Figure 5 The results of the effects of Penicillium breviscompactum Q-9 on the growth of pepper plants are shown in Figure 2. Figure 5 A is the comparison of pepper plant height between the Q-9 treatment group and the blank control group. Figure 5 B is the comparison of pepper plant height between the Q-9 treatment group and the blank control group. Figure 5 C is the comparison of dry weight of pepper plants between the Q-9 treatment group and the blank control group. DETAILED DESCRIPTION

[0024] The preferred embodiments of the present invention are described below with reference to the accompanying drawings. It should be understood that the preferred embodiments described herein are only used to illustrate and explain the present invention, and are not used to limit the present invention.

[0025] Example 1 Isolation and Identification of Penicillium brevi-compactum Q-9 Strain

[0026] 1) Isolation and purification of strains

[0027] Weigh 10 g of garlic rhizosphere soil and add it into a triangular flask containing 90 mL of sterile water. Incubate it in a shaking incubator at 25°C for 30 min, then dilute it 10-fold to 10 -1 , 10 -2 0.1 mL of the dilution solution was evenly spread on a PDA plate supplemented with streptomycin sulfate (100 ppm), and cultured in an inverted incubator at 28°C for 1-3 days, with regular observation. When suspected colonies grew in the culture dish, they were immediately picked and transferred to a fresh PDA plate for numbering and preservation. Purification was performed using the mycelial end method, and the following results were obtained: Figure 1 The PDA plate composition is: 200 g / L potato, 20 g / L glucose, and 20 g / L agar.

[0028] 2) Identification of strains

[0029] The genomic DNA of the isolated and purified strain was extracted and amplified by PCR using the following two pairs of primers:

[0030] ① Primer pairs for ITS:

[0031] Upstream primer ITS1: 5'-TCCGTAGGTGAACCTG CGG-3' (SEQ ID NO. 1),

[0032] Downstream primer ITS4: 5'-TCCTCCGCTTATTGATATGC-3' (SEQ ID NO. 2),

[0033] ② Primer pair for β-tubulin:

[0034] Upstream primer BT2a: 5'-GGTAAC CAAATC GGT GCT GCTTTC-3' (SEQ ID NO. 3),

[0035] Downstream primer Bt2b: 5′-ACC CTCAGT GTAGTGACC CTT GGC-3′ (SEQ ID NO. 4).

[0036] The amplified sequence was compared to the NCBI database using BLAST, revealing the highest similarity to Penicillium brevi-compactum. Combined with morphological identification, the strain was designated Penicillium brevi-compactum and named Q-9. The accession number is CGMCC No. 41153; the depositor is the General Microbiology Center of the China Culture Collection Administration; the address is No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, 100101, China; the date of deposit is April 11, 2024.

[0037] The Penicillium breviscompactum Q-9 strain was cultured on glucose potato agar medium PDA for 5 days. Figure 1 As shown, this strain can fill a 9-cm-diameter Petri dish. Q-9 Penicillium breviscapus colonies are bluish-green, fast-growing, densely velvety, with a smooth or rough surface, some with distinct radial grooves, and white colony margins. The hyphae are septate, the conidiophores are branched and septate, and the conidia are nearly round.

[0038] Example 2 Detection of the Growth-Promoting Effect of Penicillium breviscompactum Q-9 Strain

[0039] Prepare the phosphate solubilization test medium as follows:

[0040] 1) Phosphate Solubilization Medium: Glucose: 10g; Ca₃(PO₄)₂: 10g; MgCl₂.6H₂O: 5g; MgSO₄.7H₂O: 0.25g; KCl: 0.2g; (NH₄)₂SO₄: 0.1g; Agar: 20g; 1 L ddH₂O. Sterilize at 121°C for 20 minutes, then pour into a Petri dish and cool until ready to use.

[0041] 2) The results showed that there was a transparent halo around the colonies on the phosphate-dissolving medium, indicating that the microorganism could secrete phosphate-dissolving enzymes.

[0042] Determination of the antagonistic ability of the Penicillium breviscompactum Q-9 strain described in Example 3 against tomato and Panax notoginseng disease pathogens

[0043] The plate confrontation method was used to co-culture Penicillium breviscompactum Q-9 with 17 common crop pathogenic fungi. The pathogens include but are not limited to plant pathogens of the genera Artemia, Alternaria, Fusarium, Colletotrichum, Helminthosporium, Alternaria, Fusarium, Downy Phytophthora, Sphaerotheca, Cladosporium, Chrysospora, Rhizoctonia, Acrocystis, and Cylindrosporium, such as tomato wilt (Fusarium oxysporumf.sp.lycopersici), tomato early blight (Alternaria solani), pepper wilt (fusarium verticillioides), cucumber black heart (Cladosporium cucumerinum), litchi downy mildew (Peronophythora litchii), grape anthracnose (Colletotrichum gloeosporioides), peanut white rot (Sclerotium rolfsii), rice sheath blight (Rhizoctonia solani), rice seedling blight (Gibberella fujikuroi), rice sesame disease (Bipolaris oryzae), wheat fusarium (Fusarium graminearum), wheat take-all (Gaeumannomyces graminis var.tritici), corn leaf spot (Exserohilum turcicum), banana leaf spot (Helminthosporium torulosum (Syd.) Ashby), melon vine blight (Didymelia-brvoniaee), and Panax notoginseng rust (Cylindrocarpon destructans).

[0044] The inhibition rates of Penicillium breviscompactum Q-9 against 17 tested pathogenic strains are shown in Table 1. The specific operation is as follows:

[0045] Inoculate a 5mm diameter cake of the test pathogen at the center of a PDA culture medium. Place four Penicillium breviscompactum Q-9 cakes around the center, approximately 3cm apart. Six replicates were performed for each treatment. A control group was inoculated with only the pathogen cake. The plates were incubated at 28°C. The control colony radius was measured until it filled two-thirds of the plate. The radius of the control colony and the colony of the pathogen at the center were then measured to calculate the inhibition rate.

[0046] Inhibition rate (%) = (control colony radius - confrontation culture colony radius) / control colony radius × 100

[0047] The results are as follows Figure 3As shown, the Penicillium breviscompactum Q-9 strain significantly inhibited the growth of common crop disease pathogens. Compared with the control group, the growth of 17 pathogens tested was significantly inhibited, with inhibition rates ranging from 39.24% to 77.32%. The highest inhibition rate against rice sheath blight (Rhizoctonia solani) was 77.32%. The Penicillium breviscompactum Q-9 strain exhibits broad-spectrum antimicrobial activity.

[0048] Table 1 Antagonistic ability of Penicillium breviscompactum Q-9 strain against 17 pathogens

[0049]

[0050]

[0051] The strain Q-9 of Penicillium breviscompactum described in Example 4 can cooperate with the broad-spectrum biocontrol bacterium Pseudomonas aeruginosa

[0052] A plate standoff experiment was used to verify whether the Penicillium breviscompactum Q-9 strain could synergize with the broad-spectrum biocontrol bacterium Pseudomonas aeruginosa YNAU-A5 (P. aeruginosa). The specific experimental procedures are as follows:

[0053] Take a Q-9 bacterial cake with a diameter of 5 mm and inoculate it in the center of the PDA culture medium. Then take 5 μl of YNAU-A5 Pseudomonas bacteria solution (beneficial bacteria preserved by the applicant's laboratory) and place it around the central bacterial cake with a distance of about 3 cm between them. Figure 4 Each group was treated with 6 replicates, and the control group was inoculated with only Q-9 bacterial cake ( Figure 4 A) Culture in a 28°C constant temperature incubator until the control strain grows to 2 / 3 of the entire culture dish, and observe whether there is any interaction between the two, and whether the type of interaction is antagonistic or synergistic.

[0054] The antagonistic ability of YNAU-A5 strain was tested. Cylindrocarpondestructans, the pathogen of Panax notoginseng rust, was randomly selected as the pathogen. A 5mm diameter pathogen cake was placed in the middle. Then 5ul of YNAU-A5 Pseudomonas aeruginosa liquid was aspirated and placed around the central pathogen cake, with a distance of about 3cm between them. The culture was cultured at an appropriate temperature to test the antagonistic ability of YNAU-A5 strain. Figure 4 C).

[0055] The results show that: Figure 4 AB plate confrontation experiment showed that the short-compact Penicillium Q-9 strain and the broad-spectrum biocontrol bacteria Pseudomonas strain YNAU-A5 ( Figure 4 C) No antagonism.

[0056] On PDA plates, the Q-9 strain of Penicillium breviscompactum and the broad-spectrum biocontrol bacterium Pseudomonas aeruginosa can work synergistically, providing new ideas and possibilities for cross-border joint control of pathogens with true bacteria.

[0057] The Penicillium brevicaulis Q-9 strain described in Example 5 can promote the growth of pepper plants

[0058] A pepper pot experiment was conducted to verify whether the Penicillium breviscompactum Q-9 strain could promote pepper growth. The specific experimental procedures are as follows:

[0059] Four pepper plants were planted in each pot, and the uniformly growing peppers were divided into two treatment groups: a Q-9 bacterial solution treatment group and a blank control group, with six replicates per treatment. Purified Q-9 plates were punched into 5 mm diameter cakes and inoculated into pre-prepared, sterilized, and cooled PDB. The cultures were shaken at 28°C and 120 rpm for five days to obtain the Q-9 bacterial solution. The peppers in the Q-9 solution treatment group were irrigated with water every other week, while the blank control group received the same amount of water. Other management procedures were followed. Biomass of the peppers in each treatment was measured one month later. Six pepper plants were randomly selected, washed, and their heights were measured. The plants were then oven-dried at low temperature. Finally, the dry weight of the peppers in each treatment was measured.

[0060] The results showed that the plant height and biomass of peppers treated with Q-9 root irrigation were significantly higher than those of blank treatment ( Figure 5 AC), indicating that Penicillium breviscompactum Q-9 strain can significantly promote the growth of pepper.

[0061] Statistical analysis showed that the peppers treated with Penicillium breviscapus Q-9 had significantly higher plant height and biomass than the blank control group. The average plant height of the peppers in the treated group increased by more than 20% compared to the control group, and the plant biomass also increased significantly (see the specific data for details). Figure 5 AC). Overall, application of Penicillium breviscompactum Q-9 effectively promoted the growth and development of pepper plants. This result is consistent with the conclusion in Example 2 that Q-9 has phosphate solubilizing activity, indicating that this strain enhances plant growth by improving soil nutrient supply and the microbial environment.

[0062] Obviously, those skilled in the art may make various changes and modifications to the present invention without departing from the spirit and scope of the present invention. Thus, if such changes and modifications fall within the scope of the claims and their equivalents, the present invention is intended to include such changes and modifications.

Claims

1. A Penicillium brevicompactum Q-9 strain, characterized by: The strain was deposited in the General Microbiology Center of China Culture Collection Administration on April 11, 2024, with the deposit number CGMCC No.41153.

2. Use of the Penicillium breviscompactum Q-9 strain according to claim 1 in preventing and controlling plant diseases.

3. Use of the Penicillium brevicaulis Q-9 strain according to claim 1 in promoting plant growth.

4. A biological agent, characterized in that: The biological preparation contains the Penicillium brevicaulis Q-9 strain according to claim 1.

5. The method for preparing the biological agent according to claim 4, characterized in that: The method comprises the following steps: culturing the Penicillium breviscompactum Q-9 strain as claimed in claim 1 to proliferate the bacteria, collecting the obtained bacteria or fermentation liquid, and adding a suitable excipient to prepare the biological preparation.

6. A microbial composition, characterized in that: The microbial composition comprises the Penicillium breviscompactum Q-9 strain and the Pseudomonas strain according to claim 1.

7. Use of the microbial composition according to claim 6 in preventing and controlling plant diseases.

8. A method for preventing and controlling plant diseases, characterized in that: An effective dose of the Penicillium brevicaulis Q-9 strain according to claim 1 is applied to plants or their growth environment to prevent and control plant diseases.

9. A method for promoting plant growth, characterized in that: An effective dose of the Penicillium brevicaulis Q-9 strain according to claim 1 is applied to plants or their growth environment to promote plant growth and development.

10. The method according to claim 8, characterized in that: The Penicillium breviscompactum Q-9 strain and Pseudomonas are jointly applied to plants or their growth environment to synergistically prevent and control plant diseases.

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