Penicillium sp. NJAU-HNF8 and application thereof

Abstract

This invention discloses a Penicillium strain, NJAU-HNF8. It also discloses the inoculant prepared from it. Furthermore, it discloses the application of the strain and its prepared inoculant in controlling Fusarium wilt and root-knot nematode disease in melons, and promoting melon plant growth. This invention isolated and screened a fungal strain, NJAU-HNF8, from soil in healthy melon-growing fields. The strain possesses both antagonistic activity against Fusarium oxysporum and the ability to kill root-knot nematodes. Plate confrontation tests showed that strain NJAU-HNF8 has a strong inhibitory effect on Fusarium oxysporum. Root-knot nematode killing tests showed that the fermentation broth of strain NJAU-HNF8 can effectively kill root-knot nematodes. Pot experiments showed that treatment with the NJAU-HNF8 inoculant not only significantly reduced the number of Fusarium oxysporum in the soil and the formation of root knots in melon plants, effectively controlling Fusarium wilt and root-knot nematode disease in melons, but also better promoted the growth of melon plants.

Description

Technical Field

[0001] This invention relates to the field of agricultural microbial technology, specifically to a Penicillium strain NJAU-HNF8 and its applications. Background Technology

[0002] Melon wilt disease (caused by Fusarium oxysporum) Fusarium oxysporum f. sp. melonis Caused by) and root-knot nematode disease (mainly caused by) Meloidogyne Fusarium oxysporum (S. sp.) and root-knot nematodes are two major soil-borne diseases that harm melon production. Fusarium oxysporum invades plants through intercellular spaces in the root epidermis and through root wounds, gradually causing blockage of the vascular bundles, hindering water and nutrient transport, and in severe cases, causing the plant to wither and die. Root-knot nematodes, on the other hand, directly damage the root system, forming root knots, impairing root function, and creating pathways for other pathogens to invade. These two diseases often occur together in melon-producing areas, especially in continuously cropped fields, leading to a severe decline in yield and quality, posing a serious challenge to the green and sustainable development of the melon industry.

[0003] Currently, the control of these two diseases mainly relies on chemical control. Although chemical control is relatively quick, it has significant drawbacks: some chemical agents are highly toxic and have high residue characteristics, and long-term and large-scale use can easily disrupt the soil micro-ecological balance, causing environmental pollution and posing risks to agricultural product safety and the health of operators; long-term use of a single chemical agent can easily lead to drug resistance in pathogens (such as Fusarium oxysporum) and root-knot nematodes, reducing the control effect; most existing chemical agents are targeted at single diseases (such as special fungicides mainly controlling wilt, and special nematicides mainly controlling nematodes), making it difficult to effectively control the mixed occurrence of wilt and root-knot nematode diseases at the same time.

[0004] Biological control, due to its environmental friendliness and low likelihood of resistance development, is considered an important alternative or supplementary strategy to chemical control. However, existing biological control technologies still have significant shortcomings: biocontrol agents commonly found in the market or in research typically only show good biocontrol potential against a single target disease (Fusarium wilt or root-knot nematode disease). In field environments where both diseases coexist, these functional agents are currently insufficient to meet the needs of integrated pest management. Therefore, developing a novel, environmentally friendly microbial agent that can effectively overcome the shortcomings of existing technologies, achieve highly efficient synergistic control of Fusarium wilt and root-knot nematode disease in melons, and is of great significance for realizing green integrated pest management of soil-borne diseases in melons and ensuring the healthy development of the melon industry. Summary of the Invention

[0005] The purpose of this invention is to screen new and highly efficient biocontrol bacteria for wilt disease and root-knot nematode disease in melons, and to provide biocontrol bacteria and novel microbial agents for the biological control of these diseases.

[0006] The objective of this invention can be achieved through the following technical solutions:

[0007] The first aspect of this invention provides a Penicillium strain NJAU-HNF8, which is classified and named as follows: Penicillium citrinum It is deposited at the China General Microbiological Culture Collection Center (CGMCC) on September 8, 2025, with accession number CGMCC NO.42184.

[0008] A second aspect of the present invention provides a fungal agent prepared from the above-mentioned Penicillium NJAU-HNF, wherein the fungal agent contains ≥1×10⁻⁶ spores. 6 The spore count / mL of the inoculum should be adjusted as needed during application.

[0009] Further, the bacterial agent is prepared by the following method: strain NJAU-HNF8 is inoculated into a liquid culture medium, including PDB medium, for liquid fermentation to obtain NJAU-HNF8 fermentation broth; the NJAU-HNF8 fermentation broth is filtered through multiple layers of gauze to remove mycelia, obtaining NJAU-HNF8 spore solution; the NJAU-HNF8 spore solution is centrifuged, the supernatant is discarded, the precipitate is resuspended in sterile water, and the spore count is adjusted to ≥1×10⁻⁶. 6 per mL.

[0010] Furthermore, the liquid fermentation conditions are: temperature of 27-30℃, rotation speed of 170-190rpm, and time of 5-7 days.

[0011] The third aspect of this invention provides the application of the above-mentioned Penicillium NJAU-HNF8 in the prevention and control of melon wilt disease and root-knot nematode disease, and in promoting the growth of melon plants.

[0012] Furthermore, the melon wilt disease is caused by Fusarium oxysporum (Fusarium oxysporum). Fusarium oxysporum f. sp. melonis )cause.

[0013] Furthermore, the root-knot nematode disease is caused by root-knot nematodes ( Meloidogyne incognita )cause.

[0014] The fourth aspect of this invention provides the application of the above-mentioned microbial agent in the prevention and control of melon wilt disease and root-knot nematode disease, and in promoting the growth of melon plants.

[0015] Furthermore, the melon wilt disease is caused by Fusarium oxysporum (Fusarium oxysporum). Fusarium oxysporum f. sp. melonis )cause.

[0016] Furthermore, the root-knot nematode disease is caused by root-knot nematodes ( Meloidogyne incognita )cause.

[0017] The beneficial effects of this invention are:

[0018] This invention isolated and screened a fungal strain NJAU-HNF8 from the soil of a healthy melon growing field. This strain possesses both antagonistic activity against Fusarium oxysporum and the ability to kill root-knot nematodes. It was identified as Penicillium citrinum. Penicillium citrinum Plate confrontation experiments showed that strain NJAU-HNF8 had a strong inhibitory effect on Fusarium oxysporum. Root-knot nematode toxicity tests showed that the fermentation broth of strain NJAU-HNF8 could effectively kill root-knot nematodes. Pot experiments showed that treatment with NJAU-HNF8 not only significantly reduced the amount of Fusarium oxysporum in the soil and the formation of root knots in melon plants, effectively controlling wilt and root-knot nematode diseases in melons, but also better promoted the growth of melon plants. This invention provides excellent biocontrol strains for the biological control of wilt and root-knot nematode diseases in melons, as well as for promoting melon growth. Attached Figure Description

[0019] Figure 1 This is a diagram showing the results of the confrontation culture between strain NJAU-HNF8 and strain NJAU-HNF12.

[0020] Figure 2 This is a diagram showing the results of the confrontation culture between strain NJAU-HNF8 and Fusarium oxysporum.

[0021] Figure 3 This is a diagram showing the results of the confrontation culture between strain NJAU-HNF12 and Fusarium oxysporum.

[0022] Figure 4 A phylogenetic tree for strain NJAU-HNF8 constructed based on the ITS gene sequence.

[0023] Figure 5 The effect of NJAU-HNF8 inoculant treatment on plant height was investigated in a pot experiment.

[0024] Figure 6 The effect of NJAU-HNF8 inoculant treatment on the fresh weight of aboveground parts was investigated in a pot experiment.

[0025] Figure 7 The effect of NJAU-HNF8 inoculant treatment on the aboveground dry weight was investigated in a pot experiment.

[0026] Figure 8 The effect of NJAU-HNF8 inoculant treatment on chlorophyll value (SPAD) was investigated in a pot experiment.

[0027] Figure 9 The effect of NJAU-HNF8 inoculant treatment on the abundance of Fusarium oxysporum in soil was investigated in a pot experiment.

[0028] Figure 10The effect of NJAU-HNF8 inoculant treatment on the number of root knots in a pot experiment.

[0029] Figure 11 Images showing the effect of the control (CK) treatment on the number of root knots in a pot experiment.

[0030] Figure 12 Images showing the effect of NJAU-HNF8 inoculant treatment on the number of root knots in a pot experiment.

[0031] Different letters in the bar chart indicate significant differences (P < 0.05).

[0032] Information on the preservation of biological materials

[0033] strain NJAU-HNF8, classified and named Penicillium citrinum Latin name Penicillium citrinum The specimen is deposited at the China General Microbiological Culture Collection Center (CGMCC), located at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences. The deposit date is September 8, 2025, and the accession number is CGMCC NO.42184. Detailed Implementation

[0034] The present invention will be further explained below with reference to embodiments and accompanying drawings. The following embodiments are for illustrative purposes only and are not intended to limit the scope of the invention.

[0035] Unless otherwise specified, the plates (petition dishes) used in the following examples are all 90 mm in diameter.

[0036] The following examples involve the pathogen of Fusarium oxysporum, which causes Fusarium wilt in cantaloupe (Hami melon). Fusarium oxysporum f. sp. melonis The sample was isolated and preserved from soils highly susceptible to Fusarium wilt in cantaloupe (Hami melon) by the Modern Agriculture and Green Production Laboratory of the Sanya Research Institute of Nanjing Agricultural University.

[0037] The following examples involve *Caenorhabditis elegans* (C. elegans). Caenorhabditis elegans ) and root-knot nematodes ( Meloidogyne incognita (Provided by Jiangsu Provincial Key Laboratory of High Technology Research on Resource Utilization of Solid Organic Waste)

[0038] The following examples involve Escherichia coli (E. coli) Escherichia coli (Provided by Jiangsu Provincial Key Laboratory of High Technology Research on Resource Utilization of Solid Organic Waste)

[0039] The following examples involve culture medium formulations:

[0040] PDB medium (g / L): 6g potato flour, 20g glucose, sterilized at 121℃ for 20min. Add 2.5w / v agar powder when preparing PDB medium.

[0041] NGM nematode growth medium (g / L): peptone 2.5g, sodium chloride 3g, calcium chloride 0.111g, magnesium sulfate 0.12g, potassium dihydrogen phosphate 3.4g, agar powder 17g, sterilized at 121℃ for 20min. Before pouring plates, add 0.005g cholesterol per liter of medium (cholesterol ethanol stock solution sterilized through a 0.22μm filter membrane).

[0042] K2 Fusarium oxysporum selective medium (g / L): D-galactose 20g, L-asparagine 2g, K2HPO4 1g, KCl 5g, MgSO4 0.5g, Fe-Na-EDTA 0.01g, agar 20g, sterilized at 121℃ for 20min. Before pouring plates, add 1g pentachloronitrobenzene, 0.5g ox bile, 0.3g streptomycin sulfate, and 1g sodium tetraborate decahydrate per liter of medium. Finally, adjust the pH of the medium to 3.8-4.0 with 10wt% phosphoric acid.

[0043] Example 1: Isolation and Screening of Functional Strains

[0044] 1. Test materials:

[0045] Soil tested: Soil from a healthy cantaloupe (Hami melon) growing field.

[0046] 2. Isolation and purification of bacterial strains

[0047] The soil suspension for the isolation of the following fungal strains was prepared as follows: 18 mL of sterile water was added to a 50 mL sterile centrifuge tube, 2 g of soil sample (the test soil mentioned above) was weighed and added to the sterile water, the shaker temperature was set to 25 °C, and the mixture was shaken at 170 rpm for 2 h to obtain the soil suspension.

[0048] Isolation and purification of fungal strains: Fungal strains were isolated using the plate dilution method. Specifically: 0.1 mL of soil suspension was added to 0.9 mL of sterile water, mixed well, and then serially diluted with sterile water to a concentration of 10⁻⁶. -2 10 -3 10 -4 10 -5 10 -6 10 -7 10 were absorbed respectively -5 10 -6 10 -7 100 μL of soil dilution was evenly spread on PDA medium plates and incubated at 28°C until single colonies appeared. Colonies with different morphologies were picked and purified for later use.

[0049] 3. Preparation of fermentation broth for bacterial strains:

[0050] Preparation of fungal fermentation broth: 5 mL of sterile water was added to a fungal purification plate that had been cultured at 28℃ for 96 h. The spores and sterile water were mixed with a sterile spreader to form a spore solution. 100 μL of the spore solution was pipetted into 100 mL of PDB medium. The culture was then incubated at 28℃ and 170 rpm for 96 h with shaking. The fermentation broth was then filtered through a 0.22 μm microporous membrane (the purpose of which is to remove the fungal cells in the fermentation broth for better observation of the nematodes).

[0051] 4. Preliminary screening of nematicidal strains:

[0052] The nematode *C. elegans* was used as a model nematode for initial screening of its nematode-killing effect. The NGM nematode plates were obtained as follows: Activated *Escherichia coli* was inoculated onto NGM nematode growth medium (15-20 mL) and cultured at 37°C for 24 h. After the *E. coli* had completely covered the surface of the NGM nematode growth medium, *C. elegans* was inoculated and cultured at 25°C for 96 h to obtain the NGM nematode plates.

[0053] First, scrape 1 / 5 of the culture medium from the NGM nematode plate into a sterile plate, add 3 mL of sterile water, and mix manually for 3 min to ensure that the Caenorhabditis elegans is fully incorporated into the sterile water. Then, pipette 100 μL, 50 μL, and 20 μL of Caenorhabditis elegans suspensions at different gradients. Observe the number of Caenorhabditis elegans in the suspensions at different gradients using an inverted microscope and calculate the average number of Caenorhabditis elegans per mL of suspension. Then, in a 96-well cell culture plate, add 50 μL of the strain's fermentation broth (fungal fermentation broth) diluted 10-fold with sterile water and 50 μL of *C. elegans* suspension (ensuring a live *C. elegans* count ≥ 50, estimated based on the average number of *C. elegans* per mL of suspension). A PDB medium treatment was set up as a control (CK, i.e., replacing 50 μL of the strain's fermentation broth diluted 10-fold with sterile water with 50 μL of PDB medium). Each treatment was repeated in triplicate. The cells were incubated statically at 25°C. After 24 hours, the *C. elegans* mortality was observed under an inverted microscope, and the corrected mortality rate was calculated using the following formula.

[0054] Caenorhabditis elegans mortality rate = (Number of Caenorhabditis elegans dies during treatment) / (Number of Caenorhabditis elegans observed during treatment) × 100%

[0055] Corrected mortality rate of *C. elegans* = (mortality rate of treated *C. elegans* - mortality rate of control *C. elegans*) / (1 - mortality rate of control *C. elegans*) × 100%

[0056] As shown in Table 1, two fungal strains with a corrected mortality rate of over 40% for *C. elegans* at 24 h were obtained and named NJAU-HNF8 and NJAU-HNF12, respectively. The two strains showed antagonism. Figure 1 ).

[0057] Table 1 Corrected mortality rate of single strains against Caenorhabditis elegans

[0058]

[0059] Note: Different letters in the table indicate significant differences (P < 0.05). The same applies below.

[0060] Example 2: Antagonistic effect of functional strains against Fusarium oxysporum

[0061] The strains NJAU-HNF8 and NJAU-HNF12 obtained in Example 1, which showed good toxicity against *C. elegans*, were respectively infected with *Fusarium oxysporum*, the pathogen causing *Fusarium wilt* in melon. Fusarium oxysporum f. sp. melonis A flat-plate confrontation test was conducted.

[0062] Confrontation test between strain NJAU-HNF8 and Fusarium oxysporum: 5 mL of sterile water was added to PDA plates containing Fusarium oxysporum cultured at 28℃ for 96 h and to PDA plates containing NJAU-HNF8. The spores and sterile water were mixed with a sterile spreader to form a spore solution. Then, 2.5 μL of the spore solution was taken from each plate and symmetrically inoculated onto new PDA plates containing both Fusarium oxysporum and NJAU-HNF8 spore solutions. The plates were then incubated at 28℃. The plates were observed every 8 h for 5 consecutive days to confirm the antagonistic effect.

[0063] Confrontation test between strain NJAU-HNB12 and Fusarium oxysporum: 5 mL of sterile water was added to PDA plates containing Fusarium oxysporum cultured at 28℃ for 96 h and to PDA plates containing NJAU-HNF12. The spores and sterile water were mixed with sterile water using a sterile spreader to prepare spore solutions. Then, 2.5 μL of the spore solutions were taken from each plate and symmetrically inoculated onto new PDA plates containing both Fusarium oxysporum and NJAU-HNF12 spore solutions. The plates were then incubated at 28℃ and observed every 8 h for 5 consecutive days to confirm the antagonistic effect.

[0064] The results showed that both strains NJAU-HNF8 and NJAU-HNB12 had an inhibitory effect on Fusarium oxysporum. Figure 2 and Figure 3 ).

[0065] Example 3: The toxic effect of functional strains on root-knot nematodes

[0066] Preparation of root-knot nematode suspension: Take well-enriched root-knot nematodes ( Meloidogyne incognita Tomato roots were cleaned by washing the surface soil with water and then disinfected with 1 w / v sodium hypochlorite for 2 minutes (this disinfection only affects the surface of the tomato roots; root-knot nematodes are inside the roots and have no impact). The roots with root knots were then chopped into pieces about 1 mm in size. Root-knot nematodes were collected using a 500-mesh and 1000-mesh sieve, and then transferred to sterile centrifuge tubes with sterile water. The nematodes were observed and counted using an inverted microscope, and live nematodes were selected for later use.

[0067] In a 96-well cell culture plate, a 150 μL confrontation system was prepared, comprising: 50 μL of root-knot nematode suspension (≥50 live root-knot nematodes), 50 μL of bacterial fermentation broth diluted 10-fold with sterile water (fungal fermentation broth, prepared as in Part 3 of Example 1), and 50 μL of sterile water; a PDB medium treatment was set up as a control (CK, i.e., replacing 50 μL of bacterial fermentation broth diluted 10-fold with sterile water with 50 μL of PDB medium diluted 10-fold with sterile water, serving as a fungal control), with three replicates for each treatment. The cells were incubated statically at 25°C, and observed under an inverted microscope after 12 h, 24 h, and 36 h to record root-knot nematode mortality. The corrected mortality rate of root-knot nematodes was calculated using the following formula.

[0068] Root-knot nematode mortality rate = (Number of root-knot nematodes that died during treatment / Number of root-knot nematodes observed during treatment) × 100%

[0069] Corrected mortality rate of root-knot nematodes = (mortality rate of treated root-knot nematodes - mortality rate of control root-knot nematodes) / (1 - mortality rate of control root-knot nematodes) × 100%

[0070] Table 2 Corrected mortality rate of root-knot nematodes by single strains

[0071]

[0072] Analysis of the experimental results in Table 2 shows that the treatment with fermentation broth containing strain NJAU-HNF8 resulted in a corrected mortality rate of 55.65% for root-knot nematodes after 36 hours. This indicates that the fermentation broth containing strain NJAU-HNF8 can effectively kill root-knot nematodes.

[0073] Example 4 Identification of Functional Strains

[0074] Strain NJAU-HNF8 was cultured on PDA medium at 28℃ for 96 h. The mycelium was bluish-green, the colonies were round with regular edges, and the spores were green or yellowish-green, easily picked up, and the mycelium was tightly bound to the medium. The ITS gene sequence of strain NJAU-HNF8 was compared with similar sequences, and a phylogenetic tree was constructed (…). Figure 4 The results showed that strain NJAU-HNF8 and Penicillium citrinum ICJ14 showed the highest homology. Based on the colony morphology characteristics of strain NJAU-HNF8 and the phylogenetic tree comparison analysis using the ITS gene sequence, strain NJAU-HNF8 was identified as *Penicillium citrinum*. Penicillium citrinum The phylogenetic tree results showed that this fungus is harmless to crops and non-pathogenic to humans and animals. The strain NJAU-HNF8 has been deposited at the China General Microbiological Culture Collection Center (CGMCC) on September 8, 2025, with accession number CGMCC NO.42184.

[0075] Example 5: Study on the effects of functional strain NJAU-HNF8 on melon plant growth, soil Fusarium oxysporum abundance, and root knot quantity using a pot experiment.

[0076] The pot experiment was conducted in the cultivation room of Nanjing Agricultural University Sanya Research Institute in Sanya City, Hainan Province, from February to April 2025. The tested melon variety was Xizhoumi 25.

[0077] The test inoculum was NJAU-HNF8. The preparation method for NJAU-HNF8 inoculum is as follows:

[0078] The activated strain NJAU-HNF8 was inoculated onto a PDA medium plate and cultured at 28℃ for 96 h to obtain a purified fungal strain plate. Approximately 5 mL of sterile water was added to the purified fungal strain plate, and the spores and sterile water were mixed thoroughly with a sterile spreader to prepare a spore suspension. 200 μL of the spore suspension was pipetted into 250 mL of PDB medium and cultured at 28℃ and 170 rpm for 7 days to obtain the NJAU-HNF8 fermentation broth. The NJAU-HNF8 fermentation broth was filtered through four layers of sterile gauze to remove mycelia, yielding the NJAU-HNF8 spore suspension. The NJAU-HNF8 spore suspension was centrifuged at 3000 rpm for 5 min, the supernatant was discarded, and the precipitate was resuspended in sterile water and the spore count was adjusted to 1 × 10⁶. 7 The NJAU-HNF8 bacterial agent was obtained by obtaining cells / mL.

[0079] In the preparation stage of NJAU-HNF8 bacterial agent, the strain was amplified and cultured in PDB medium. After the culture was completed, the supernatant was discarded by centrifugation, the precipitate was resuspended in sterile water and the spore number was adjusted. Therefore, PDB medium will not interfere with the functional effect of the final bacterial agent.

[0080] The tested soil was inoculated with Fusarium oxysporum (Fusarium oxysporum) Fusarium oxysporum f. sp. melonis ) and root-knot nematodes ( Meloidogyne incognita Soils with high incidence of diseases.

[0081] The pot experiment included two treatments: 1) CK, treated with sterile water; and 2) NJAU-HNF8, treated with NJAU-HNF8 inoculant. Each treatment had six replicates, with one pot per replicate.

[0082] The pot experiment was conducted in a greenhouse with the temperature set at 30℃ for 16 hours with light on, followed by 26℃ for 8 hours with light off, and an air humidity of 60%. The melon seedlings were self-bred; after germination, the seeds were cultivated for 7 days, and seedlings of uniform growth were transplanted into pots (approximately 11cm high, 16.5cm in diameter at the rim, and 10.5cm in diameter at the bottom). The test soil and vermiculite were mixed at a mass ratio of 43:7, and each pot was filled to approximately 2cm below the rim. One melon seedling was transplanted into each pot, and the pots were watered until water seeped out from the bottom. Subsequent watering followed standard practices. One week after transplanting, 50mL of a fungicide was applied to each pot via root irrigation, while the control (CK) received an equal volume of sterile water. Forty days after transplanting, six pots from each treatment were collected for the determination of plant height, aboveground fresh weight, aboveground dry weight, chlorophyll value (SPAD), soil Fusarium oxysporum count, and root knot count. The methods for measuring each indicator are as follows:

[0083] Plant height: Use a measuring tape to measure from the soil surface of the melon plant to the top of the main stem.

[0084] Fresh weight of above-ground parts: The above-ground parts of the melon plant are cut off and weighed to obtain the fresh weight of the above-ground parts.

[0085] Aboveground dry weight: The aboveground parts of the melon plant were placed in an oven and dried at 65°C until constant weight, and then weighed to obtain the aboveground dry weight.

[0086] Chlorophyll value (SPAD): The chlorophyll value (SPAD) of the largest leaf of the melon plant was measured using a handheld chlorophyll meter (SPAD-502PIU).

[0087] Soil Fusarium oxysporum count: The count of Fusarium oxysporum in the rhizosphere soil of melon plants was determined using the dilution spread count method. The specific steps are as follows: The melon plant was cut off at the soil surface. The aboveground part of the melon plant was used for the determination of aboveground fresh weight and aboveground dry weight. The underground part of the melon plant, along with the rhizosphere soil, was carefully dug out as a whole, keeping the root system as intact as possible. Loose soil and large particles attached to the root surface were gently shaken off, and stones and other impurities were removed. 20 mL of sterile water was placed in a 50 mL sterile centrifuge tube. The treated melon roots were placed in the centrifuge tube and shaken at 25℃ and 170 rpm for 1 hour until there was no obvious soil attached to the root surface. The roots were then removed with sterile forceps to obtain the rhizosphere soil suspension. 0.9 mL of sterile water and 0.1 mL of rhizosphere soil suspension were added to a 1.5 mL sterile centrifuge tube, mixed well, and then serially diluted with sterile water to 10⁻⁶. -2 10 -3 10 -4 10-5 10 were absorbed respectively -3 10 -4 10 -5 100 μL of soil dilution was spread evenly on K2 Fusarium oxysporum selective medium plates using a spreading stick and incubated at 28°C until single colonies appeared. The number of Fusarium oxysporum was then counted to obtain the number of Fusarium oxysporum (unit: CFU / mL rhizosphere soil suspension).

[0088] Root knot count: Place the melon roots after measuring the Fusarium oxysporum count on a transparent petri dish or graph paper, and count them one by one: each visible swelling (regardless of size) is counted as one root knot. Use tweezers and a counter to count them one by one.

[0089] like Figures 5 to 12 As shown, the plant height, aboveground fresh weight, aboveground dry weight, and chlorophyll value of the NJAU-HNF8 inoculant treatment were all higher than those of the CK treatment, with the aboveground dry weight showing a significant difference. The number of Fusarium oxysporum in the soil and the number of root knots in the roots of the NJAU-HNF8 inoculant treatment were significantly lower than those of the CK treatment. Therefore, the NJAU-HNF8 inoculant treatment not only significantly reduces the number of Fusarium oxysporum in the soil and the formation of root knots in melon plants, but also better promotes the growth of melon plants.

Claims

1. A Penicillium strain NJAU-HNF8, characterized in that, Its classification is named Penicillium citrinum It is deposited at the China General Microbiological Culture Collection Center (CGMCC) on September 8, 2025, with accession number CGMCC NO.42184.

2. The inoculum prepared from Penicillium NJAU-HNF according to claim 1, characterized in that, The bacterial agent contains ≥1×10⁶ spores. 6 per mL.

3. The microbial agent according to claim 2, characterized in that, The microbial agent is prepared by the following method: strain NJAU-HNF8 is inoculated into a liquid culture medium, including PDB medium, for liquid fermentation to obtain NJAU-HNF8 fermentation broth; the NJAU-HNF8 fermentation broth is filtered through multi-layer gauze to remove mycelia, yielding NJAU-HNF8 spore solution; the NJAU-HNF8 spore solution is centrifuged, the supernatant is discarded, the precipitate is resuspended in sterile water, and the spore count is adjusted to ≥1×10⁻⁶. 6 per mL.

4. The microbial agent according to claim 3, characterized in that, The liquid fermentation conditions are: temperature 27-30℃, rotation speed 170-190rpm, and time 5-7 days.

5. The application of Penicillium NJAU-HNF8 as described in claim 1 in the prevention and control of wilt disease and root-knot nematode disease in melons and the promotion of melon plant growth.

6. The application according to claim 5, characterized in that, The melon wilt disease is caused by Fusarium oxysporum (Fusarium oxysporum). Fusarium oxysporum f. sp. melonis )cause.

7. The application according to claim 5, characterized in that, The root-knot nematode disease is caused by root-knot nematodes ( Meloidogyne incognita )cause.

8. The application of the microbial agent according to any one of claims 2-4 in the prevention and control of melon wilt disease, root-knot nematode disease, and promotion of melon plant growth.

9. The application according to claim 8, characterized in that, The melon wilt disease is caused by Fusarium oxysporum (Fusarium oxysporum). Fusarium oxysporum f. sp. melonis )cause.

10. The application according to claim 8, characterized in that, The root-knot nematode disease is caused by root-knot nematodes ( Meloidogyne incognita )cause.

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