Penicillium brevicompactum and its application in biological control and growth promotion

Abstract

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

Description

Technical Field

[0001] This invention relates to the field of microbiology, specifically to a non-pathogenic Penicillium short-dense mold and its application in plant control and growth promotion. Background Technology

[0002] Penicillium short-dense Penicillium brevi-compactum The Q-9 strain, recruited from sulfur-containing compounds in garlic, possesses growth-promoting properties and can effectively antagonize some pathogens causing serious diseases in Solanaceae and Panax notoginseng. It can also synergize with the broad-spectrum biocontrol bacterium *Pseudomonas*. P. aeruginosa ).

[0003] Tomatoes, belonging to the Solanaceae family and the Solanum genus, are an important vegetable crop that has rapidly developed in my country's agricultural industry, making my country one of the major planting and producing countries. Rich in vitamins, lycopene, and other nutrients, tomatoes are popular due to their ability to supplement human bodily functions. However, besides being affected by natural conditions such as region and weather, tomato cultivation is also inevitably affected by diseases, which are diverse and can cause significant yield losses in severe cases. Early blight and wilt are important global diseases affecting tomatoes, with high incidence rates and substantial yield losses. Fusarium oxysporum (Fusarium oxysporum) is another example. Fusarium oxysporum Fusarium wilt caused by chemical control is characterized by large damage and difficulty in prevention and control. Currently, chemical control can easily cause environmental pollution and pesticide residues. Long-term use of chemical agents can also easily induce drug resistance in pathogens and food safety issues.

[0004] Sanqi Panax notoginseng Panax notoginseng is a perennial plant cultivated under no-till shade in the Araliaceae family. It thrives in warm, humid conditions and is a renowned Chinese medicinal herb, considered a unique and rare medicinal plant in China. Due to its extremely high medicinal value, it is revered as "invaluable" and "the divine herb of the South." Due to the specific limitations of its variety and growing environment, there are few reports of research on it abroad. It possesses effects such as promoting blood circulation, dispersing blood stasis, reducing swelling and relieving pain, anti-oxidation, and anti-aging. Continuous cropping obstacles refer to the imbalance of soil microbial communities when the same species or family of crops is continuously planted in the same land, even under normal management conditions. This leads to reduced germination rates, increased pests and diseases, and ultimately affects product quality. Root rot is one of the most serious diseases affecting Panax notoginseng, accounting for more than 70% of all diseases, and can reduce yield by 5% to 70% or even cause total crop failure. Furthermore, the soil often requires an interval of more than 10 years before replanting. These problems seriously affect the sustainable cultivation and yield of Panax notoginseng, therefore, effective measures are urgently needed to solve this problem.

[0005] Microbial synergy refers to the phenomenon where two or more microorganisms interact in a specific environment, thereby jointly influencing their growth, metabolism, or biological functions. This synergistic relationship is widespread in nature, such as in soil, oceans, within plants and animals, and in the human gut microbiota. Two microorganisms form complex ecological networks through nutrient exchange, metabolite secretion, and signal transduction, thus significantly impacting environmental adaptation, resource utilization, and the expression of specific functions. Research on microbial synergy not only reveals the ecological relationships between microorganisms but also provides an important applied foundation for agriculture, medicine, and environmental science.

[0006] Microorganisms can secrete plant hormones, which can induce resistance-promoting effects, hyperparasitism, and enhance the plant's ability to develop resistance. However, most current biocontrol bacteria exhibit either strong antagonistic effects but poor broad-spectrum control, or vice versa. Therefore, developing biocontrol bacteria that possess both resistance and broad-spectrum control is another challenge in biological control. Summary of the Invention

[0007] To solve the above-mentioned technical problems, the present invention provides the following technical solution: Firstly, in a first aspect, this invention provides a *Penicillium brevicompactum* strain Q-9. This strain, identified as *Penicillium brevicompactum*, is non-pathogenic and has been deposited at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC No. 41153 (deposit date: April 11, 2024). This strain grows rapidly on potato dextrose agar (PDA), forming blue-green, velvety colonies with white edges, and is capable of secreting lysozyme and other active substances.

[0008] A second aspect of this invention provides the application of the aforementioned Penicillium short-dense strain Q-9 in the control of plant diseases. This strain exhibits antagonistic activity against various 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 preparing a biocontrol agent from strain Q-9 and applying it to plants or soil, the growth of pathogens can be effectively inhibited, reducing the occurrence of diseases.

[0009] A third aspect of this invention provides the application of the aforementioned Penicillium breve strain Q-9 in promoting plant growth. This strain significantly promotes plant growth and development by secreting plant growth stimulants and enhancing soil nutrient availability (such as dissolving insoluble phosphorus). For example, after treatment with strain Q-9, the plant height and biomass of pepper plants were significantly higher than those of the untreated control.

[0010] A fourth aspect of this invention provides a biological agent containing the *Penicillium breve* strain Q-9 and a method for preparing the same. This biological agent uses strain Q-9 as the active ingredient and can be formulated into suspensions, wettable powders, and other dosage forms. The preparation method includes: culturing and amplifying strain Q-9 to obtain a large number of bacterial cells or spores; if necessary, the fermentation broth or bacterial cells can be mixed with a carrier to prepare the product. This biological agent can be used for soil treatment, seed coating, or plant spraying to achieve disease control and crop growth promotion.

[0011] A fifth aspect of this invention provides a technical solution for the combined control of plant diseases by *Penicillium breve* strain Q-9 and another broad-spectrum biocontrol bacterium, *Pseudomonas aeruginosa*. By applying strain Q-9 in combination with *Pseudomonas aeruginosa* YNAU-A5, the control spectrum can be further broadened and the control effect enhanced. The two strains exhibit no antagonistic effect under co-culture conditions, demonstrating symbiotic compatibility and producing a synergistic inhibitory effect on the target pathogen, thus providing a new combination strategy for the biological control of crop diseases.

[0012] Compared with the prior art, the present invention has significant beneficial effects: 1. Broad-spectrum and highly effective disease control: The Penicillium breve Q-9 strain exhibits strong antagonistic activity against a variety of plant pathogenic fungi. Experimental results show that the inhibition rate against 17 common plant pathogens tested ranged from 39.24% to 77.32%, with the highest inhibition rate of 77.32% against Rhizoctonia solani, the pathogen causing rice sheath blight. Therefore, this strain can be used to control fungal diseases in various crops, overcoming the limitation of single biocontrol agents in terms of their limited control spectrum.

[0013] 2. Significant Plant Growth-Promoting Effect: The *Penicillium breve* Q-9 strain can secrete beneficial enzymes such as phosphatase, promoting the release of insoluble phosphorus from the soil and improving the plant's nutrient absorption and utilization rate. Pot experiments showed that after treatment with the Q-9 strain, the plant height and biomass of pepper plants were significantly higher than the control group (untreated plants), proving that this strain has a significant growth-promoting effect. Therefore, the application of the Q-9 strain can promote crop growth and increase crop yield while preventing disease.

[0014] 3. Good safety and compatibility: The Q-9 strain originates from plant rhizosphere soil, is non-pathogenic, safe, and environmentally friendly, posing no harm to crops or 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-species synergistic use of fungi and bacteria provides new possibilities for biological control, simultaneously leveraging the advantages of multiple microorganisms to improve the stability and broad-spectrum effectiveness of control measures.

[0015] In summary, the Penicillium breve Q-9 strain provided by this invention has both broad-spectrum antibacterial and growth-promoting effects, and can be used to develop highly efficient biological control agents to reduce dependence on chemical pesticides. It has significant agricultural application value and promising prospects for promotion. Attached Figure Description

[0016] The accompanying drawings are provided to further illustrate the invention and form part of the specification. They are used in conjunction with embodiments of the invention to explain the invention and do not constitute a limitation thereof. In the drawings: Figure 1 The morphological characteristics of Penicillium brevicornuate strain Q-9 on PDA medium are shown, where a and b are colony morphology, and c is hyphae and conidia morphology. Figure 2 A transparent halo was generated for lysophosphatase production in Penicillium brevicornum strain Q-9; Figure 3 The plates are plates after 7 days of confrontation culture of three pathogens of Penicillium breve Q-9 strain. The top three images are the control group, and the bottom three images are plates with Penicillium breve Q-9 strain added respectively. Figure 4 A: Colony diagram of Q-9 on a PDA; Figure 4 B:Q-9 can synergize with pseudomonocytes; Figure 4 C: Pseudomonas antagonizes the pathogen causing rust in Panax notoginseng and destroys Cyclospora ( Cylindrocarpon destructans ); Figure 5 The results show the effects of treatment with Penicillium brevis strain Q-9 on the growth of pepper plants, among which... Figure 5 A represents a comparison of the chili plant height between the Q-9 treatment group and the blank control group. Figure 5 B represents a comparison of the plant height of pepper plants in the Q-9 treatment group and the blank control group. Figure 5 C represents the comparison of the dry weight of chili plants between the Q-9 treatment group and the blank control group. Detailed Implementation

[0017] The preferred embodiments of the present invention will be described below with reference to the accompanying drawings. It should be understood that the preferred embodiments described herein are for illustration and explanation only and are not intended to limit the present invention.

[0018] Example 1 Penicillium brevi-compactum Isolation and identification of Penicillium brevichalum strain Q-9 1) Isolation and purification of strains Weigh 10 g of garlic rhizosphere soil and add it to an Erlenmeyer flask containing 90 mL of sterile water. Incubate at 25 °C with shaking for 30 min, then perform a 10-fold serial dilution to 10⁻⁶. -1 10 -20.1 mL of the diluted solution was evenly spread onto a PDA plate containing streptomycin sulfate (100 ppm). The plate was incubated upside down at 28 ℃ for 1-3 days, with regular observation. Once suspected colonies appeared in the culture dish, they were immediately picked and transferred to fresh PDA plates for labeling and preservation. Purification was performed using the hyphal tip method to obtain the desired product. Figure 1 The pure culture shown is illustrated. The PDA plate composition is: potato 200 g / L, glucose 20 g / L, and agar 20 g / L.

[0019] 2) Identification of strains Genomic DNA was extracted from the purified strain and amplified by PCR using the following two pairs of primers: ① Primer pairs for ITS: Upstream primer ITS1: 5'-TCCGTAGGTGAACCTG CGG-3' (SEQ ID NO.1) Downstream primer ITS4: 5'-TCCTCCGCTTATTGATATGC-3' (SEQ ID NO.2), ② Primer pair for β-tubulin: Upstream primer BT2a: 5'-GGT AAC CAA ATC GGT GCT GCT TTC-3' (SEQ ID NO.3). Downstream primer Bt2b: ​​5'-ACC CTC AGT GTA GTG ACC CTT GGC-3' (SEQ ID NO.4).

[0020] The amplified sequences were compared with those in the NCBI database. BLAST comparison showed the highest similarity to *Penicillium brevi-compactum*. Combined with morphological identification, the strain was named *Penicillium brevi-compactum*, and the classification name was confirmed as *Penicillium brevi-compactum*, with the strain name Q-9. The accession number is CGMCC No. 41153; the depositary institution is the China General Microbiological Culture Collection Center; the depositary address is No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, 100101, China; the deposit date is April 11, 2024.

[0021] The *Penicillium brevicornu* strain Q-9 was cultured on glucose potato agar (PDA) medium for 5 days, as... Figure 1As shown, this strain can completely cover a 9 cm diameter petri dish. The Q-9 short-dense Penicillium colonies are blue-green, grow rapidly, are dense and velvety, with smooth or rough surfaces, some showing distinct radial grooves, and have white edges. The hyphae are septate, the conidiophores are branched and septate, and the conidia are nearly round.

[0022] Example 2: Detection of the growth-promoting effect of Penicillium breve Q-9 strain The following steps are taken to prepare the phosphate-soluble detection medium: 1) Phosphate-solubilizing medium: Glucose: 10g; Ca3(PO4)2: 10g; MgCl2·6H2O: 5g; MgSO4·7H2O: 0.25g; KCl: 0.2g; (NH4)2SO4: 0.1g; Agar: 20g; 1L ddH2O. Sterilize at 121℃ for 20 minutes, then pour into petri dishes and cool before use.

[0023] 2) The results showed that on the phosphate-solubilizing medium, there was a transparent halo around the colony, indicating that the microorganism could secrete phosphate lysozyme.

[0024] Example 3: Determination of the antagonistic ability of the Penicillium short-dense strain Q-9 against pathogens causing diseases of tomatoes and Panax notoginseng. A plate confrontation method was used to co-culture *Penicillium brevesense* strain Q-9 with 17 common crop pathogenic fungi. These pathogens included, but were not limited to, plant pathogens of the genera *Amanita*, *Alternaria*, *Fusarium*, *Anthracis*, *Helicobacter*, *Alternaria*, *Fusarium*, *Phytophthora*, *Gnaphalium*, *Cladosporium*, *Rhizoctonia*, *Rhizoctonia*, and *Cyclospora*, such as those found in tomato wilt (*Pseudomonas*). Fusarium oxysporum f.sp.lycopersici ) 、 Early blight of tomatoes ( Alternaria solani ) 、 Chili peppers wither ( fusarium verticillioides ) 、 Black-hearted cucumbers Cladosporium cucumerinum ) 、 Lychee downy mildew ( Peronophythora litchii ) 、 Grape anthrax ( Colletotrichum gloeosporioides ) 、 Peanut white mold disease ( Sclerotium rolfsii ), rice sheath blight ( Rhizoctonia solani ) 、 Rice seedlings ( Gibberella fujikuroi ) 、 Rice sesame disease ( Bipolaris oryzae ) 、 wheat gibberellin Fusarium graminearum ), total eclipse of wheat ( Gaeumannomyces graminis var.tritici ) 、 Large Spotted Corn Exserohilum turcicum ) 、 Banana leaf spots ( Helminthosporium torulosum(Syd.)Ashby )、 Cucurbit vine blight ( Didymelia-brvoniaee ) 、 rust of Panax notoginseng ( Cylindrocarpon destructans ).

[0025] The inhibition rates of Penicillium breve Q-9 strain against 17 tested pathogen strains are shown in Table 1. The specific procedures are as follows: One 5 mm diameter mycelial disc of the tested pathogen was inoculated into the center of a PDA medium. Four more mycelial discs of *Penicillium brevesii* strain Q-9 were placed around the center disc, approximately 3 cm apart. Each treatment was repeated six times. The control group consisted of only mycelial discs inoculated with the pathogen. The cultures were incubated at 28 ℃. Once the control strain had covered two-thirds of the culture dish, the colony radius of the control strain and the colony radius of the opposing central pathogen were measured, and the inhibition rate was calculated.

[0026] Inhibition rate (%) = (Control colony radius - Confrontation culture colony radius) / Control colony radius × 100 The results are as follows Figure 3 As shown, the *Penicillium breve* strain Q-9 significantly inhibited the growth of common crop pathogens. Compared with the control group, the growth of 17 tested pathogens was significantly inhibited, with inhibition rates ranging from 39.24% to 77.32%. Among them, it significantly inhibited the growth of rice sheath blight (…). Rhizoctonia solani The antagonistic inhibition rate of ) was the highest, reaching 77.32%. The Penicillium breve Q-9 strain has broad-spectrum antibacterial activity.

[0027] Table 1. Antagonistic ability of Penicillium breve Q-9 strain against 17 pathogenic fungi. Example 4 The Penicillium short-dense strain Q-9 can interact with the broad-spectrum biocontrol bacterium Pseudomonas aeruginosa ( P. aeruginosa Collaboration A plate confrontation experiment was used to verify whether Penicillium breve Q-9 strain could interact with the broad-spectrum biocontrol bacterium Pseudomonas YNAU-A5 ( P. aeruginosa Collaboration. Specific experimental procedures are as follows: Take one Q-9 bacterial disc with a diameter of 5 mm and inoculate it in the center of PDA medium. Then, take 5 μL of YNAU-A5 Pseudomonas bacterial suspension (beneficial bacteria preserved in the applicant's laboratory) and place it around the center bacterial disc, with a distance of about 3 cm between each piece. Figure 4 B). Each treatment was repeated 6 times, with the control group inoculated only with Q-9 mycelium cake (…). Figure 4 A) was cultured in a constant temperature incubator at 28 ℃. After the control group strain had grown to 2 / 3 of the entire culture dish, we observed whether there was any interaction between the two strains and whether the interaction was antagonistic or synergistic.

[0028] Antagonistic ability of YNAU-A5 strain was determined, and the pathogen of Panax notoginseng rust was randomly selected. Cylindrocarpon destructans As a pathogen, a 5 mm diameter bacterial cake of pathogen was placed in the center, and then 5 μL of YNAU-A5 Pseudomonas bacterial solution was placed around the central pathogen bacterial cake, with a distance of about 3 cm between each layer. After incubation at a suitable temperature, the antagonistic ability of the YNAU-A5 strain was tested. Figure 4 C).

[0029] The results show that: Figure 4 AB plate confrontation experiment shows that Penicillium breve Q-9 strain and broad-spectrum biocontrol fungus Pseudomonas strain YNAU-A5 ( Figure 4 C) No antagonistic effect.

[0030] On PDA plates, *Penicillium breve* strain Q-9 and the broad-spectrum biocontrol bacterium *Pseudomonas* (… P. aeruginosa They can work synergistically, providing new ideas and possibilities for cross-species collaboration between eubacteria and bacteria to jointly control pathogens.

[0031] Example 5: The Penicillium short-dense strain Q-9 can promote the growth of pepper plants. A pot experiment was conducted on chili peppers to verify whether the Penicillium brevis strain Q-9 could promote chili pepper growth. The specific experimental procedure is as follows: Four chili pepper plants were planted in each pot, and the plants were divided into two groups: a Q-9 bacterial solution treatment group and a blank control group, with each treatment replicated six times. Purified Q-9 plates were broken into 5 mm diameter mycelial cakes and inoculated into pre-sterilized and cooled PDB (polydiol) substrate. The plants were incubated at 28 ℃ and 120 rpm for 5 days to obtain the Q-9 bacterial solution. The roots of the Q-9 bacterial solution-treated chili pepper plants were drenched once a week, while the blank control group was drenched with the same amount of water. All other management procedures were the same. Biomass was measured after one month. Six chili pepper plants were randomly selected, washed, and their height was measured. The plants were then dried in a low-temperature oven, and the dry weight of the chili peppers in each treatment was weighed.

[0032] The results showed that the plant height and biomass of peppers treated with Q-9 root irrigation were significantly higher than those of the control group. Figure 5 AC) indicates that the Penicillium short-dense strain Q-9 can significantly promote the growth of peppers.

[0033] Statistical analysis showed that the pepper group treated with Penicillium brevis strain Q-9 had significantly higher average plant height and biomass than the control group. Specifically, the average plant height in the treatment group increased by more than 20% compared to the control group, and plant biomass also showed a significant increase (see details). Figure 5(AC). Overall, application of Penicillium breve Q-9 strain effectively promoted the growth and development of pepper plants. This result is consistent with the conclusion in Example 2 that the Q-9 strain has phosphorus-solubilizing activity, indicating that this strain enhances plant growth performance by improving soil nutrient supply and the microbial environment.

[0034] Obviously, those skilled in the art can make various modifications and variations to this invention without departing from its spirit and scope. Therefore, if these modifications and variations fall within the scope of the claims of this invention and their equivalents, this invention also intends to include these modifications and variations.

Claims

1. Penicillium brevicornuum ( Penicillium brevicompactum The application of strain Q-9 in the prevention and control of plant diseases is characterized by: The strain was deposited on April 11, 2024, at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC No. 41153. The plant diseases mentioned are selected from the following: tomato wilt, tomato early blight, pepper wilt, cucumber black heart, lychee downy mildew, grape anthracnose, peanut white mold, rice sheath blight, rice bakanae disease, rice sesame disease, wheat scab, wheat take-all, corn leaf blight, banana leaf spot, cucurbit vine blight, or Panax notoginseng rust rot.

2. Penicillium brevicornuum ( Penicillium brevicompactum The application of strain Q-9 in promoting plant growth is characterized by: The strain was deposited on April 11, 2024, at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC No. 41153, and the plant is pepper.

3. A method for preventing and controlling plant diseases, characterized in that: Applying an effective dose of the *Penicillium brevicornum* strain Q-9 of claim 1 to plants or their growing environment to prevent and control plant diseases selected from the following: tomato wilt, tomato early blight, pepper wilt, cucumber black heart, lychee downy mildew, grape anthracnose, peanut white mold, rice sheath blight, rice bakanae disease, rice sesame disease, wheat scab, wheat take-all, corn leaf blight, banana leaf spot, cucurbit vine blight, or *Panax notoginseng* rust rot.

4. A method for promoting plant growth, characterized in that: Applying an effective dose of the Penicillium short-dense strain Q-9 of claim 2 to a plant or its growth environment to promote plant growth and development, said plant being a pepper.

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