A compound microbial agent containing penicillium citrinum and trichoderma guizhouense, and a preparation method and application thereof

Abstract

This invention discloses a compound fungal agent containing Penicillium citrinum and Trichoderma guizhouense, wherein the Penicillium citrinum is Penicillium citrinum (… Penicillium citrinum NJAU-T106, the Guizhou Trichoderma is Guizhou Trichoderma ( Trichoderma guizhouense NJAU4742. This invention also discloses its preparation method and its application in promoting banana plant growth, increasing banana yield, and controlling banana wilt disease. This invention screened Penicillium citrinum (NJAU4742) from banana disease-suppressing soil to obtain a strain with good biocontrol and growth-promoting effects. Penicillium citrinum The study used NJAU-T106 and innovatively combined it with Trichoderma guiyuanensis NJAU4742 to construct a synergistic system of Penicillium citrinum and Trichoderma guiyuanensis. This effectively reduced the incidence of banana wilt disease while better promoting banana plant growth and increasing banana yield, thereby driving the green and sustainable development of the banana industry.

Description

Technical Field

[0001] This invention relates to the field of agricultural microbial technology, specifically to a compound microbial agent containing Penicillium citrinum and Trichoderma guizhouense, its preparation method, and its application. Background Technology

[0002] Banana (scientific name: Musa nana Bananas (Lour.) are perennial herbaceous plants native to tropical and subtropical Southeast Asia. They have become an important food and economic crop in developing tropical and subtropical countries worldwide. China is one of the earliest countries in the world to cultivate and grow bananas on a large scale, and is now the world's second-largest banana producer, with major production areas including Guangdong, Hainan, Guangxi, Fujian, and Yunnan. However, with the expansion of banana cultivation, the fungus *Fusarium oxysporum* (…) has become a major cause of banana disease. Fusarium oxysporum f. sp. Cubense Fusarium wilt (FOC) caused by bananas has become a major obstacle to the sustainable development of the banana industry.

[0003] Banana wilt is a devastating soil-borne vascular disease caused by Fusarium oxysporum. This pathogen infects the banana root and vascular system, hindering water and nutrient transport, leading to wilting and death of the plant. Bananas are susceptible throughout their growth cycle, and the disease spreads widely through soil, irrigation water, and farm implements. Once Fusarium oxysporum contaminates banana plantation soil, traditional methods are insufficient for complete eradication in the short term. Chemical control not only has limited effectiveness but also easily causes environmental pollution, pesticide residues, and pathogen resistance. With the development of green agriculture, biological control has become the most promising control strategy due to its environmental friendliness and sustainability.

[0004] Currently, the control effect of single biocontrol strains is often limited, while complex microbial systems can significantly enhance antibacterial ability and promote plant growth through the synergistic effect between different strains. Summary of the Invention

[0005] The purpose of this invention is to provide a compound fungal agent containing Penicillium citrinum and Trichoderma guizhouense, its preparation method and application, in order to overcome the shortcomings of the prior art.

[0006] To achieve the above objectives, the present invention adopts the following technical solution:

[0007] The first aspect of this invention provides a compound fungal agent containing Penicillium citrinum and Trichoderma guizhouense, wherein the Penicillium citrinum is Penicillium citrinum (… Penicillium citrinum NJAU-T106, deposited at the China General Microbiological Culture Collection Center (CGMCC) on September 8, 2025, with accession number CGMCC No. 42183; the *Trichoderma guiyuanensis* mentioned is *Trichoderma guiyuanensis* (…). Trichoderma guizhouenseNJAU4742 is deposited at the China General Microbiological Culture Collection Center (CGMCC) on April 11, 2016, with accession number CGMCCNo.12166.

[0008] Furthermore, the compound fungal agent containing Penicillium citrinum and Trichoderma guizhouense is prepared by the following steps:

[0009] Penicillium citrinum NJAU-T106 was inoculated into liquid culture medium for liquid fermentation to obtain a culture broth. The culture broth was filtered to remove mycelia, obtaining Penicillium citrinum NJAU-T106 spore suspension. The Penicillium citrinum NJAU-T106 spore suspension was centrifuged, the supernatant was discarded, the precipitate was resuspended in sterile water, and the spore concentration was adjusted to ≥1×10⁻⁶ with sterile water. 6 1 spore / mL, to obtain Penicillium citrinum NJAU-T106 inoculum;

[0010] Trichoderma guiyuan NJAU4742 was inoculated into a liquid culture medium for liquid fermentation to obtain a culture broth. The culture broth was filtered to remove mycelia, obtaining Trichoderma guiyuan NJAU4742 spore suspension. The Trichoderma guiyuan NJAU4742 spore suspension was centrifuged, the supernatant was discarded, the precipitate was resuspended in sterile water, and the spore concentration was adjusted to ≥1×10⁻⁶ with sterile water. 6 The inoculum of *Trichoderma guiyuan* NJAU4742 was obtained by spores / mL.

[0011] The compound fungal agent containing Penicillium citrinum NJAU-T106 and Trichoderma guiyang NJAU4742 was obtained by mixing equal volumes of these two fungal agents.

[0012] Furthermore, the liquid culture medium used for the liquid fermentation culture of Penicillium citrinum NJAU-T106 and Trichoderma guizhouense NJAU4742 included PDB liquid culture medium.

[0013] Furthermore, the liquid fermentation conditions for Penicillium citrinum NJAU-T106 and Trichoderma guizhouense NJAU4742 were: temperature 28-30℃, rotation speed 170-200 rpm, and fermentation time 5-7 days.

[0014] The second aspect of this invention provides a method for preparing a compound fungal agent containing *Penicillium citrinum* and *Trichoderma guizhouense*, wherein the *Penicillium citrinum* is *Penicillium citrinum* (… Penicillium citrinum NJAU-T106, deposited at the China General Microbiological Culture Collection Center (CGMCC) on September 8, 2025, with accession number CGMCC No. 42183; the *Trichoderma guiyuanensis* mentioned is *Trichoderma guiyuanensis* (…). Trichoderma guizhouenseNJAU4742 is deposited at the China General Microbiological Culture Collection Center (CGMCC) on April 11, 2016, with accession number CGMCC No. 12166.

[0015] The preparation of the compound fungal agent containing Penicillium citrinum and Trichoderma guizhouense includes the following steps:

[0016] Penicillium citrinum NJAU-T106 was inoculated into liquid culture medium for liquid fermentation to obtain a culture broth. The culture broth was filtered to remove mycelia, obtaining Penicillium citrinum NJAU-T106 spore suspension. The Penicillium citrinum NJAU-T106 spore suspension was centrifuged, the supernatant was discarded, the precipitate was resuspended in sterile water, and the spore concentration was adjusted to ≥1×10⁻⁶ with sterile water. 6 1 spore / mL, to obtain Penicillium citrinum NJAU-T106 inoculum;

[0017] Trichoderma guiyuan NJAU4742 was inoculated into a liquid culture medium for liquid fermentation to obtain a culture broth. The culture broth was filtered to remove mycelia, obtaining Trichoderma guiyuan NJAU4742 spore suspension. The Trichoderma guiyuan NJAU4742 spore suspension was centrifuged, the supernatant was discarded, the precipitate was resuspended in sterile water, and the spore concentration was adjusted to ≥1×10⁻⁶ with sterile water. 6 The inoculum of *Trichoderma guiyuan* NJAU4742 was obtained by spores / mL.

[0018] The compound fungal agent containing Penicillium citrinum NJAU-T106 and Trichoderma guiyang NJAU4742 was obtained by mixing equal volumes of these two fungal agents.

[0019] Furthermore, the liquid culture medium used for the liquid fermentation culture of Penicillium citrinum NJAU-T106 and Trichoderma guizhouense NJAU4742 included PDB liquid culture medium.

[0020] Furthermore, the conditions for liquid fermentation culture of Penicillium citrinum NJAU-T106 and Trichoderma guizhouense NJAU4742 were: temperature 28-30℃, rotation speed 170-200rpm, and fermentation culture time 5-7 days.

[0021] The third aspect of this invention provides the application of the above-mentioned compound microbial agent containing Penicillium citrinum and Trichoderma guizhouense in promoting banana plant growth, increasing banana yield, and preventing banana wilt disease.

[0022] Furthermore, during application, the compound microbial agent containing Penicillium citrinum and Trichoderma guizhouense is applied once during the seedling stage and once during the vegetative growth stage of banana plants, with 0.8-1L applied to each banana plant each time by root irrigation.

[0023] The beneficial effects of this invention are:

[0024] This invention screened Penicillium citrinum from banana disease-suppressing soil to obtain a strain with good biocontrol and growth-promoting effects. Penicillium citrinum The study used NJAU-T106 and innovatively combined it with Trichoderma guiyuanensis NJAU4742 to construct a synergistic system of Penicillium citrinum and Trichoderma guiyuanensis. This effectively reduced the incidence of banana wilt disease while better promoting banana plant growth and increasing banana yield, thereby driving the green and sustainable development of the banana industry. Attached Figure Description

[0025] Figure 1 Photograph of a single culture of the pathogen causing banana wilt.

[0026] Figure 2 Photographs showing the confrontation between strain NJAU-E11 and the pathogen causing banana wilt.

[0027] Figure 3 Photographs showing the confrontation between strain NJAU-G20 and the pathogen causing banana wilt.

[0028] Figure 4 Photographs showing the confrontation between strain NJAU-H18 and the pathogen causing banana wilt.

[0029] Figure 5 Photographs showing the confrontation between strain NJAU-T106 and the pathogen causing banana wilt.

[0030] Figure 6 This is a graph showing the inhibition results of various strains against the pathogen causing banana wilt.

[0031] Figure 7 This is a photograph of the colony morphology (front view) of strain NJAU-T106.

[0032] Figure 8 This is a photograph of the colony morphology of strain NJAU-T106 (back side).

[0033] Figure 9 Phylogenetic tree diagram of strain NJAU-T106 constructed based on 16S rRNA sequence.

[0034] Figure 10 This is a graph showing the inhibition results of different treatments on the pathogen of banana wilt in Example 3.

[0035] Figure 11 Phenotypic photographs showing the effects of different treatments on banana seedling growth in a pot experiment.

[0036] Figure 12 Effects of different treatments on banana seedling growth in pot experiments - aboveground fresh weight.

[0037] Figure 13 Effects of different treatments on banana seedling growth in pot experiments - aboveground dry weight.

[0038] Figure 14 Effects of different treatments on banana seedling growth in pot experiments - chlorophyll content.

[0039] Figure 15 To investigate the effects of different treatments on the number of banana wilt pathogens in the soil during a pot experiment.

[0040] Figure 16 The effect of different treatments on banana plant growth in a field experiment - plant height increase.

[0041] Figure 17 The effect of different treatments on banana plant growth in a field experiment - stem diameter increase.

[0042] Figure 18 To investigate the effects of different treatments on the yield of banana plants in a field experiment.

[0043] Figure 19 To investigate the effects of different treatments on banana yield per acre in a field experiment.

[0044] Figure 20 To investigate the effects of different treatments on the incidence of banana wilt disease in a field trial.

[0045] Figure 21 To investigate the effects of different treatments on the number of banana wilt pathogens in the soil during a field experiment.

[0046] Different lowercase letters in the above bar charts indicate significant differences between treatments (p<0.05).

[0047] Information on the preservation of biological materials

[0048] NJAU-T106, classified as Penicillium citrinum. 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. 42183.

[0049] NJAU4742, classified as *Trichoderma guiyuan*. Trichoderma guizhouense Latin name Trichoderma guizhouense It 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, on April 11, 2016, with accession number CGMCC NO.12166. Detailed Implementation

[0050] The following examples are provided to better understand the present invention, but should not be construed as limiting the invention. Unless otherwise specified, the experimental methods in the following examples are conventional methods. Unless otherwise specified, the experimental materials used in the following examples were purchased from conventional biochemical reagent stores.

[0051] Unless otherwise specified, the raw materials used in the following examples are as follows:

[0052] The seedling substrate is Xingxing Xiangnong brand special substrate produced by Jiangsu Xingnong Substrate Technology Co., Ltd., with product number 161102G0097N.

[0053] PDB liquid culture medium (1L): 6.0g potato extract powder, 20.0g glucose, deionized water to a final volume of 1L, sterilize at 115℃ for 30min.

[0054] 1 / 10 PDA solid culture medium (1L): 0.6g potato extract powder, 2.0g glucose, deionized water to a final volume of 1L, then add 20.0g agar powder, sterilize at 115℃ for 30min.

[0055] PDA solid medium (1L): 6.0g potato extract powder, 20.0g glucose, deionized water to a final volume of 1L, then add 20.0g agar powder, sterilize at 115℃ for 30min.

[0056] K2 solid medium (1L): 1.0g dipotassium hydrogen phosphate, 0.5g potassium chloride, 0.5g magnesium sulfate, 0.01g sodium iron EDTA, 2.0g L-asparagine, 20g D-galactose, and deionized water to a final volume of 1L. Adjust the pH to 3.8±0.2 with 10wt% phosphoric acid, then add 16.0g agar powder. Sterilize at 115℃ for 30min. Cool to approximately 60℃, and add 1.0g pentachloronitrobenzene (75% WP), 0.5g ox bile, 1.0g sodium tetraborate, and 0.3g streptomycin sulfate per liter of medium.

[0057] Unless otherwise specified, the plates (petition dishes) used in the following examples are all 90 mm in diameter.

[0058] Example 1: Isolation and Screening of Functional Strains

[0059] 1. Test materials:

[0060] The pathogen of banana wilt was tested as follows: Fusarium oxysporum, a specialized race of Fusarium oxysporum var. cucumeritum, type 4 (…). Fusarium oxysporum f. sp. cubense race 4, FOC4), provided by Jiangsu Provincial Key Laboratory of High Technology Research on Resource Utilization of Solid Organic Waste.

[0061] 2. Isolation of strains

[0062] Rhizosphere soil samples were collected from healthy banana plants in fields affected by Fusarium wilt. These samples were air-dried, crushed, and sieved through a 10-mesh sieve. 10g of the soil sample was weighed and poured into a 90mL Erlenmeyer flask containing 4-6 glass beads of sterile physiological saline. The flask was shaken at 30℃ and 170rpm for 30 minutes to obtain an initial soil suspension. This initial soil suspension was then serially diluted 10-fold to prepare a 10-fold concentration gradient. -2 10 -3 10 -4 10 -5 10 -6 Soil suspensions at different concentration gradients. 100 μL of soil suspensions at different concentration gradients were plated onto 1 / 10 PDA solid medium plates, with each gradient replicated three times. The plates were then incubated at 28°C for 3-7 days. Once colonies appeared, single colonies of different fungi were selected and purified based on their color, morphology, and size to obtain purified strains.

[0063] 3. Screening of strains

[0064] The preserved *Fusarium wilt* pathogen FOC4 was inoculated onto PDA solid medium plates and cultured at 28°C until the mycelium completely covered the plate and produced a large number of spores. Sterile water was added, and the surface of the colonies was gently scraped with a spreader to obtain a *Fusarium wilt* pathogen FOC4 bacterial suspension. The *Fusarium wilt* pathogen FOC4 bacterial suspension was filtered through four layers of sterile gauze to remove the mycelium, obtaining a *Fusarium wilt* pathogen FOC4 spore suspension. The spore concentration in the *Fusarium wilt* pathogen FOC4 spore suspension was determined by hemocytocyte count, and the spore concentration was adjusted to 1 × 10⁻⁶ with sterile water. 6 1 spore / mL. Spore solutions of each purified strain were obtained using the same method described above (spore concentration 1×10⁻⁶). 6 (spores / mL).

[0065] The inhibitory ability of each purified strain against the growth of *Fusarium wilt* pathogen of banana was determined using a plate confrontation test. 1 μL of *Fusarium wilt* spore suspension (FOC4) was pipetted into the center of a PDA solid medium plate, incubated for 30 min, and then cultured at 28°C for 24 h. Subsequently, 1 μL of purified strain spore suspension was inoculated approximately 2 cm from the center of the PDA solid medium plate on the right side, while the corresponding left side remained uninoculated. A control treatment was set up where neither the right nor left side was inoculated. Each treatment was repeated three times. After incubation at 28°C for 5 days, the colony radius of *Fusarium wilt* FOC4 was measured for each treatment, and the inhibition rate was calculated to determine the inhibitory ability of each purified strain against the growth of *Fusarium wilt* pathogen. The inhibition rate was calculated as follows: Inhibition rate = (Coronary radius of control treatment - Colony radius of purified strain treatment) / Colony radius of control treatment × 100%. After screening hundreds of strains, the results are as follows: Figures 1 to 6 As shown, strains NJAU-T106, NJAU-H18, NJAU-G20, and NJAU-E11 exhibited good plate confrontation effects, with inhibition rates of 41.67%, 32.0%, 30.0%, and 7.4% against the growth of the banana wilt pathogen, respectively. Among them, strain NJAU-T106 showed the most significant inhibitory effect on the growth of the banana wilt pathogen.

[0066] 4. Identification of strains

[0067] After strain NJAU-T106 was cultured on PDA solid medium plates at 28°C for 2 weeks, as follows: Figure 7 (front) and Figure 8 (Reverse side) As shown, the colonies are powdery, round, relatively smooth, with a raised center and neat edges, and no obvious aerial hyphae. They are initially lemon yellow, later covered with a dense brownish-brown spore layer, slightly darker in the center, and generally flat without obvious wrinkles. The reverse side of the colonies is light brown to yellowish-brown, with concentric rings, gradually fading at the edges, and no pigment diffusion into the culture medium. The color difference between the front and back sides is significant. A phylogenetic tree constructed based on the 16S rDNA gene sequence of strain NJAU-T106 (as shown in SEQ ID NO.1) is as follows: Figure 9 As shown, strain NJAU-T106 and Penicillium citrinum P1.21 showed the highest homology, reaching 99.82%.

[0068] Based on the phylogenetic tree comparison analysis of the colony morphology and 16S rDNA gene sequence of strain NJAU-T106, strain NJAU-T106 was identified as *Penicillium citrinum*. Penicillium citrinum It was deposited on September 8, 2025, at the China General Microbiological Culture Collection Center (CGMCC), with accession number CGMCCNO.42183.

[0069] Example 2 Preparation of bacterial agent

[0070] Strain NJAU-T106 was inoculated onto PDA solid medium plates and cultured at 28°C until the mycelium completely covered the plate and produced abundant spores. The spore suspension was obtained by rinsing with sterile water. 0.1 mL of the spore suspension was inoculated into a 250 mL Erlenmeyer flask containing 100 mL of PDB liquid medium and cultured at 28°C and 170 rpm for 7 days to obtain the culture medium. The culture medium was filtered through four layers of sterile gauze to remove the mycelium, yielding NJAU-T106 spore solution. The spore concentration was determined by hemocytocyte counting to be ≥1×10⁻⁶. 7 1 spore / mL. Centrifuge the NJAU-T106 spore solution at 5000 rpm for 10 min, discard the supernatant, resuspend the precipitate in sterile water, and adjust the spore concentration to 1×10⁶ spores / mL with sterile water. 6 1 spore / mL, yielding NJAU-T106 bacterial agent.

[0071] Strain NJAU4742 was inoculated onto PDA solid medium plates and cultured at 28°C until the mycelium completely covered the plate and produced abundant spores. The spore suspension was obtained by rinsing with sterile water. 0.1 mL of the spore suspension was inoculated into a 250 mL Erlenmeyer flask containing 100 mL of PDB liquid medium and cultured at 28°C and 170 rpm for 7 days to obtain the culture medium. The culture medium was filtered through four layers of sterile gauze to remove the mycelium, yielding NJAU4742 spore solution. The spore concentration was determined by hemocytocyte counting to be ≥1×10⁻⁶. 7 1 spore / mL. Centrifuge the NJAU4742 spore solution at 5000 rpm for 10 min, discard the supernatant, resuspend the precipitate in sterile water, and adjust the spore concentration to 1×10⁻⁶ spores / mL with sterile water. 6 1 spore / mL, yielding NJAU4742 inoculum.

[0072] Example 3: Inhibition of banana wilt pathogen by the combination of strain NJAU-T106 and strain NJAU4742.

[0073] The banana wilt pathogen FOC4 was inoculated onto PDA solid medium plates and cultured at 28°C until the mycelium completely covered the plate and produced a large number of spores. The spore suspension was obtained by rinsing with sterile water. 0.1 mL of the spore suspension was inoculated into a 250 mL Erlenmeyer flask containing 100 mL of PDB liquid medium and cultured at 28°C and 170 rpm for 7 days to obtain the culture medium. The culture medium was filtered through four layers of sterile gauze to remove the mycelium, yielding the spore solution of the banana wilt pathogen FOC4.

[0074] A 6-well plate micro-soil culture system was used. The soil (collected from a continuous cropping park with typical symptoms of banana wilt disease, where Brazilian bananas have been continuously planted for more than 10 years; same as the soil tested in Example 4) was sterilized at 121°C for 1 hour and then cooled for later use.

[0075] A total of 4 processes are set up, namely:

[0076] 1) Apply sterile water as a blank control, recorded as CK treatment;

[0077] 2) Apply NJAU-T106 bacterial agent (prepared in Example 2, spore concentration 1×10⁻⁶). 6 (spores / mL), denoted as NJAU-T106 treatment;

[0078] 3) Apply NJAU4742 inoculant (prepared in Example 2, spore concentration 1×10⁻⁶). 6 (spores / mL), denoted as NJAU4742 treatment;

[0079] 4) Apply NJAU-T106 inoculant (prepared in Example 2, spore concentration 1×10⁻⁶). 6 (spores / mL) and NJAU4742 inoculum (prepared in Example 2, spore concentration of 1×10⁻⁶) 6 A mixture of equal volumes of microbial agents (spores / mL) is designated as NJAU-T106+NJAU4742 treatment.

[0080] Each treatment was designed with 3 replicates, with 1 replicate per well. 10 g (dry weight) of sterilized soil was added to each well for each treatment. In the control (CK) treatment, 2 mL of sterile water was added to each well, and in the NJAU-T106 treatment, 2 mL of NJAU-T106 inoculant (spore concentration 1×10⁻⁶) was added to each well. 6 In the NJAU4742 treatment, 2 mL of NJAU4742 bacterial agent (spore concentration of 1×10⁶ spores / mL) was applied to each well. 6 In the NJAU-T106+NJAU4742 treatment, 2 mL of NJAU-T106 bacterial agent (spore concentration of 1×10⁶ / mL) was applied to each well. 6 (spores / mL) and NJAU4742 inoculum (spore concentration of 1×10⁻⁶) 6 A mixed inoculant was prepared by mixing equal volumes of soil and liquid (spores / mL). After application, the soil and liquid were thoroughly mixed and incubated at 28°C for 7 days. Each treatment was then treated with a spore suspension of FOC4, the pathogen of Fusarium wilt of banana (before application, the FOC4 spore suspension was centrifuged at 5000 rpm for 10 min, the supernatant was discarded, the precipitate was resuspended in sterile water, and the spore concentration was adjusted to 1×10⁻⁶ using a hemocytometer). 7200 μL of FOC4 spore solution (1 × 10⁶ spores / mL) of banana wilt pathogen was slowly and evenly applied to each well. 7 (Spores / mL), then add 100 μL of sterile water to maintain humidity. 14 days after inoculation with the banana wilt pathogen FOC4, 0.25 g of soil was taken from each well to extract total DNA, and the amount of banana wilt pathogen in the soil was quantified by real-time quantitative PCR.

[0081] like Figure 10 As shown, the number of Fusarium wilt pathogens in bananas treated with NJAU-T106+NJAU4742 was significantly lower than that in the CK, NJAU-T106, and NJAU4742 treatments, indicating that the NJAU-T106+NJAU4742 treatment had a more significant inhibitory effect on Fusarium wilt pathogens in bananas.

[0082] Example 4: Effect of synergistic treatment of strains NJAU-T106 and NJAU4742 on potted control of banana wilt disease.

[0083] The pot experiment was conducted in the greenhouse of Nanjing Agricultural University Sanya Research Institute in Sanya City, Hainan Province. The greenhouse conditions were set as follows: temperature 30℃, relative humidity 80%, and light / dark cycle 16h / 8h.

[0084] The test material was Brazilian banana ( Musa AAA Cavendish tissue culture seedlings, conforming to the NY / T 357-2007 standard for Class II tissue culture seedlings, are a susceptible variety of banana wilt disease and were provided by Hainan Tropical Crops Research Institute Seed Industry Technology Co., Ltd.

[0085] The test soil was collected from a continuously cropped orchard exhibiting typical symptoms of banana wilt disease, where Brazilian bananas had been continuously grown for over 10 years. The test soil and quartz sand were mixed evenly at a volume ratio of 3:1 to obtain a mixed soil. The mixed soil was then placed into plastic basins (160 mm high, 98 mm top diameter, 61 mm bottom diameter), with each basin containing 600 g (dry weight) of the mixed soil for later use.

[0086] Prepare sterile seedling substrate and seedling trays (50 cells per tray, 4.5cm diameter, 4.5cm depth, and 2cm bottom diameter per cell). Fill the seedling trays with the sterile seedling substrate. After washing the root culture medium from the Brazilian banana tissue culture seedlings, transplant them into the seedling trays, one seedling per cell. Cover with a suitable film, provide shade, and maintain moisture. Place them in a greenhouse for cultivation. Use a bottom watering method for water management. First, thoroughly water the seedling substrate from the top until water drips from the bottom of the seedling tray, ensuring the substrate reaches saturation. Then, place the seedling trays in a tray filled with water, maintaining a water level of 1-2cm in the tray. Check and replenish water every 2-3 days to maintain the water level. Regularly irrigate with sterile modified Hogland's nutrient solution (Jiangsu Aidisheng Biotechnology Co., Ltd., product number: ADS101M0). In the early stage of transplanting, use 1 / 4 of the working solution (pH 5.8-6.0). After new roots sprout, switch to 1 / 2 of the working solution (pH 5.8-6.0) to ensure the banana seedlings have the necessary nutrients.

[0087] Once the banana seedlings have grown 3-5 true leaves, select seedlings of uniform growth and transplant them into plastic pots filled with mixed soil, one seedling per pot, for a potted plant experiment. During the potted plant experiment, water thoroughly every 2-3 days.

[0088] The banana seedlings transplanted into plastic pots were divided into four treatments:

[0089] 1) Apply sterile water as a blank control, recorded as CK treatment;

[0090] 2) Apply NJAU-T106 bacterial agent (prepared in Example 2, spore concentration 1×10⁻⁶). 6 (spores / mL), denoted as NJAU-T106 treatment;

[0091] 3) Apply NJAU4742 inoculant (prepared in Example 2, spore concentration 1×10⁻⁶). 6 (spores / mL), denoted as NJAU4742 treatment;

[0092] 4) Apply NJAU-T106 inoculant (prepared in Example 2, spore concentration 1×10⁻⁶). 6 (spores / mL) and NJAU4742 inoculum (prepared in Example 2, spore concentration of 1×10⁻⁶) 6 A mixture of equal volumes of microbial agents (spores / mL) is designated as NJAU-T106+NJAU4742 treatment.

[0093] Each treatment had four replicates, with one plastic pot per replicate. After transplanting, the banana seedlings were allowed to stabilize for one week. In the control (CK) treatment, each seedling was treated with 60 mL of sterile water via root drenching. In the NJAU-T106 treatment, each seedling was treated with 60 mL of NJAU-T106 inoculant (spore concentration 1×10⁻⁶) via root drenching.6 In the NJAU4742 treatment, 60 mL of NJAU4742 inoculant (spore concentration of 1×10⁶ spores / mL) was applied to each banana seedling via root irrigation. 6 In the NJAU-T106+NJAU4742 treatment, 60 mL of NJAU-T106 inoculant (spore concentration of 1×10⁶ / mL) was applied to each banana seedling via root irrigation. 6 (spores / mL) and NJAU4742 inoculum (spore concentration of 1×10⁻⁶) 6 A mixed inoculum agent of equal volume (spores / mL) was prepared. After culturing for another 2 weeks, each treatment was then treated with FOC4 spores of the banana wilt pathogen (the FOC4 spores were prepared according to the method described in Example 3, and before application, the FOC4 spores were centrifuged at 5000 rpm for 10 min, the supernatant was discarded, the precipitate was resuspended in sterile water, and the spore concentration was adjusted to 1×10⁻⁶ using a hemocytometer). 7 6 mL of FOC4 spore solution (1×10⁶ spores / mL) was applied to each pot of banana seedlings via root irrigation. 7 (Spores / mL). After two more weeks of culture, three banana seedlings were randomly selected from each treatment. The aboveground fresh weight, aboveground dry weight, and chlorophyll content (SPAD value) of the banana seedlings were measured. The pathogen of banana wilt in the potted soil was quantified by real-time quantitative PCR, and the growth status of banana seedlings in each treatment was compared.

[0094] The methods for measuring each indicator are as follows:

[0095] Determination of fresh weight of aboveground parts: Remove the banana seedlings completely from the plastic pot, gently shake off the loose soil attached to the roots, rinse the surface of the banana seedlings with deionized water to remove any remaining soil, and then gently absorb the surface moisture with absorbent paper. Separate the aboveground parts (including pseudostems and leaves) from the root system at the root-stem junction with scissors, and immediately weigh the aboveground part sample using an analytical balance to obtain the fresh weight of the aboveground parts.

[0096] Determination of dry weight of aerial parts: After weighing the fresh weight of the aerial parts, place them into a marked paper bag and put them in a constant temperature drying oven. First, fix the green at 105℃ for 30 minutes, then lower the temperature to 75℃ and continue drying for 48 hours until the aerial parts sample reaches constant weight. Take out the aerial parts sample, place it in a desiccator to cool to room temperature, and weigh it using an analytical balance to obtain the dry weight of the aerial parts.

[0097] Chlorophyll content (SPAD value): The chlorophyll content was measured using a chlorophyll meter. The upper, middle and lower leaves of the banana seedlings were selected for measurement, and the average of the measured values ​​was taken as the final result.

[0098] like Figure 11As shown, the banana seedlings treated with NJAU-T106+NJAU4742 had longer and denser root systems, stronger stems and leaves, and more expansive leaves. In terms of root development and stem and leaf growth, they were superior to the NJAU-T106 treatment, NJAU4742 treatment, and CK treatment.

[0099] like Figure 12-14 As shown, the aboveground fresh weight, aboveground dry weight, and chlorophyll content (SPAD value) of banana seedlings treated with NJAU-T106+NJAU4742 were significantly higher than those treated with CK, NJAU-T106, and NJAU4742. The aboveground fresh weight, aboveground dry weight, and chlorophyll content (SPAD value) of banana seedlings treated with NJAU-T106 and NJAU4742 were also higher than those treated with CK, with the aboveground fresh weight showing a significant difference. Figure 15 As shown, the number of banana wilt pathogens in the soil treated with NJAU-T106+NJAU4742 was significantly lower than that in the CK, NJAU-T106, and NJAU4742 treatments. The number of banana wilt pathogens in the soil treated with NJAU-T106 and NJAU4742 was lower than that in the CK treatment, with the NJAU4742 treatment showing a significant difference.

[0100] It is evident that both NJAU-T106 and NJAU4742 inoculants significantly promoted banana seedling growth and, to some extent, inhibited the number of banana wilt pathogens in the soil. Synergistic treatment with NJAU-T106 and NJAU4742 inoculants further significantly promoted banana seedling growth and inhibited the number of banana wilt pathogens in the soil.

[0101] Example 5: Field control effect of synergistic treatment of strains NJAU-T106 and NJAU4742 on banana wilt disease.

[0102] The field trial was conducted at the banana plantation of Hainan Yikang Ecological Agriculture Development Co., Ltd. in Fushan Town, Chengmai County, Hainan Province. The plantation had been cultivating Brazilian bananas for over ten years, with a Fusarium wilt incidence rate exceeding 40%. The basic soil conditions at the experimental site were: pH approximately 5.48, available phosphorus 47.71 mg / kg, available potassium 162.54 mg / kg, available nitrogen 181.58 mg / kg, and organic matter 19.46 g / kg. The tested banana variety was Brazilian banana, with seedlings two months old, provided by a banana nursery in Daji Village, Fushan Town, Chengmai County, Hainan Province. The experiment included four treatments:

[0103] 1) Apply sterile water as a blank control, recorded as CK treatment;

[0104] 2) Apply NJAU-T106 bacterial agent (prepared in Example 2, spore concentration 1×10⁻⁶). 6(spores / mL), denoted as NJAU-T106 treatment;

[0105] 3) Apply NJAU4742 inoculant (prepared in Example 2, spore concentration 1×10⁻⁶). 6 (spores / mL), denoted as NJAU4742 treatment;

[0106] 4) Apply NJAU-T106 inoculant (prepared in Example 2, spore concentration 1×10⁻⁶). 6 (spores / mL) and NJAU4742 inoculum (prepared in Example 2, spore concentration of 1×10⁻⁶) 6 A mixture of equal volumes of solvent (spores / mL) is referred to as NJAU-T106+NJAU4742 treatment.

[0107] The field trial employed a randomized block design, with four treatments, three replicates per treatment, and a total of twelve plots. Each plot had an area of ​​20 m². 2 (12.5m long, 1.6m wide), with 8 banana plants planted in each plot, and the plots are randomly arranged within the area group.

[0108] The banana plants were treated once each during the seedling stage (30 days after transplanting) and the vegetative growth stage (90 days after transplanting). The procedures for each treatment were as follows: In the control (CK) treatment, 1L of sterile water was applied to each banana plant via root drenching; in the NJAU-T106 treatment, 1L of NJAU-T106 inoculant (spore concentration of 1×10⁻⁶) was applied to each banana plant via root drenching. 6 In the NJAU4742 treatment, 1 L of NJAU4742 inoculant (spore concentration of 1×10⁶ spores / mL) was applied to each banana plant via root irrigation. 6 In the NJAU-T106+NJAU4742 treatment, 1L of NJAU-T106 inoculant (spore concentration of 1×10⁻⁶ / mL) was applied to each banana plant via root irrigation. 6 (spores / mL) and NJAU4742 inoculum (spore concentration of 1×10⁻⁶) 6 A mixed solvent containing equal volumes of spores per mL was prepared. All other field management practices remained consistent across treatments.

[0109] Plant height and stem diameter were measured at 60 and 180 days after transplanting for each treatment. Plant height was measured using a steel tape measure as the vertical distance from the highest leaf to the ground, and stem diameter was measured using calipers as the diameter of the pseudostem 10 cm above the ground. The increase in biological traits (plant height and stem diameter) during the rapid growth period (60–180 days) was expressed as the difference between the values ​​measured at 180 days and 60 days after transplanting. To ensure the representativeness and consistency of the results, five representative banana plants with uniform growth were selected from all replicate plots for each treatment. The samples included plants with varying degrees of natural disease in the field (including healthy plants and mildly diseased plants), without excluding diseased plants. During the experiment, all selected plants were uniformly tagged, and the same batch of tagged plants was continuously tracked and measured at each measurement time point.

[0110] After the banana plants reached maturity, three uniformly growing and representative banana plants (a total of nine plants per treatment) were selected from each replicate plot. Whole bunches of bananas were harvested from each plant and weighed. The yield per banana plant and the yield per acre were calculated using the following formulas.

[0111] Banana yield per plant (kg / plant) = Total weight of 3 banana plants / 3

[0112] Banana yield per mu (kg / mu) = Yield per banana plant × Total number of banana plants in the plot × 666.67m 2 / Total area of ​​Banana Community

[0113] The occurrence of Fusarium wilt was investigated throughout the entire growth period of banana plants. Plants exhibiting typical Fusarium wilt symptoms, such as poor new leaf development, wilting and yellowing of older leaves, and browning and cracking at the stem base, were identified as diseased plants. The number of diseased plants in each treatment was recorded, and the incidence rate was calculated using the following formula:

[0114] Incidence rate (%) = (Number of diseased plants in the plot / Total number of plants in the plot) × 100%

[0115] 120 days after transplanting, soil samples were collected from five representative banana plants of uniform growth (the same plants selected for measuring plant height and stem diameter growth) in each replicate plot for each treatment. Soil samples were collected from the top 0-30 cm of soil inside the drip line of the banana plants. After thorough mixing of soil samples from the same location, approximately 300g of soil was retained as a single soil sample using the quartering method. The pathogen of banana wilt was then quantified in the soil samples using real-time quantitative PCR.

[0116] like Figure 16As shown, during the rapid growth period of banana plants (60–180 days after transplanting), different treatments had varying effects on plant height growth. The NJAU-T106+NJAU4742 treatment resulted in the highest plant height growth, significantly superior to other treatments. Compared to the control (CK) treatment, the growth was 82.22% higher, compared to the NJAU-T106 treatment, 20.59% higher, and compared to the NJAU4742 treatment, 12.33% higher. The NJAU-T106 and NJAU4742 treatments were also significantly superior to the CK treatment, increasing height by 51.11% and 62.22%, respectively.

[0117] like Figure 17 As shown, during the rapid growth period of banana plants (60–180 days after transplanting), different treatments had varying effects on stem diameter growth. The NJAU-T106+NJAU4742 treatment resulted in the highest stem diameter growth, significantly superior to other treatments. Compared to the control (CK) treatment, the growth was increased by 42.70%, compared to the NJAU-T106 treatment by 21.19%, and compared to the NJAU4742 treatment by 18.22%. The NJAU-T106 and NJAU4742 treatments were also significantly superior to the CK treatment, increasing the growth by 17.74% and 20.70%, respectively.

[0118] like Figure 18 and Figure 19 As shown, the yield per plant and yield per acre of the NJAU-T106+NJAU4742 treatment were significantly higher than those of other treatments. The yield per plant of the NJAU-T106+NJAU4742 treatment increased by 100.94% compared to the CK treatment, by 30.89% compared to the NJAU-T106 treatment, and by 13.33% compared to the NJAU4742 treatment. The yield per acre of the NJAU-T106+NJAU4742 treatment increased by 108.02% compared to the CK treatment, by 30.90% compared to the NJAU-T106 treatment, and by 24.29% compared to the NJAU4742 treatment. The yield per plant and yield per acre of the NJAU-T106 and NJAU4742 treatments were also significantly better than those of the CK treatment, with yield per plant increasing by 58.87% and 83.48%, respectively, and yield per acre increasing by 58.91% and 67.36%, respectively.

[0119] like Figure 20As shown, compared with the control (CK) treatment, the NJAU-T106, NJAU4742, and NJAU-T106+NJAU4742 treatments all significantly reduced the incidence of Fusarium wilt in banana plants. There was no significant difference in the incidence of Fusarium wilt among the three treatments (NJAU-T106, NJAU4742, and NJAU-T106+NJAU4742), with the NJAU-T106+NJAU4742 treatment exhibiting the lowest incidence.

[0120] like Figure 21 As shown, compared with the control (CK) treatment, the NJAU-T106, NJAU4742, and NJAU-T106+NJAU4742 treatments all significantly reduced the number of Fusarium wilt pathogens in the soil. There was no significant difference in the number of Fusarium wilt pathogens in the soil among the three treatments (NJAU-T106, NJAU4742, and NJAU-T106+NJAU4742), with the NJAU-T106+NJAU4742 treatment showing the lowest number of Fusarium wilt pathogens in the soil.

[0121] Based on the results of pot experiments and field trials, the combined treatment of NJAU-T106 and NJAU4742 inoculants significantly improved the growth and yield of banana plants compared to either treatment alone, and also effectively suppressed the occurrence of banana wilt disease.

Claims

1. A compound microbial agent containing Penicillium citrinum and Trichoderma guizhouense, characterized in that, The *Penicillium citrinum* is *Penicillium citrinum* (… Penicillium citrinum NJAU-T106, deposited at the China General Microbiological Culture Collection Center (CGMCC) on September 8, 2025, with accession number CGMCC No. 42183; the *Trichoderma guiyuanensis* mentioned is *Trichoderma guiyuanensis* (…). Trichoderma guizhouense NJAU4742 is deposited at the China General Microbiological Culture Collection Center (CGMCC) on April 11, 2016, with accession number CGMCC No. 12166.

2. The compound fungal agent containing Penicillium citrinum and Trichoderma guizhouense according to claim 1, characterized in that, The compound fungal agent containing Penicillium citrinum and Trichoderma guizhouense is prepared by the following steps: Penicillium citrinum NJAU-T106 was inoculated into liquid culture medium for liquid fermentation to obtain a culture broth. The culture broth was filtered to remove mycelia, obtaining Penicillium citrinum NJAU-T106 spore suspension. The Penicillium citrinum NJAU-T106 spore suspension was centrifuged, the supernatant was discarded, the precipitate was resuspended in sterile water, and the spore concentration was adjusted to ≥1×10⁻⁶ with sterile water. 6 1 spore / mL, to obtain Penicillium citrinum NJAU-T106 inoculum; Trichoderma guiyuan NJAU4742 was inoculated into a liquid culture medium for liquid fermentation to obtain a culture broth. The culture broth was filtered to remove mycelia, obtaining Trichoderma guiyuan NJAU4742 spore suspension. The Trichoderma guiyuan NJAU4742 spore suspension was centrifuged, the supernatant was discarded, the precipitate was resuspended in sterile water, and the spore concentration was adjusted to ≥1×10⁻⁶ with sterile water. 6 The inoculum of *Trichoderma guiyuan* NJAU4742 was obtained by spores / mL. The compound fungal agent containing Penicillium citrinum NJAU-T106 and Trichoderma guiyang NJAU4742 was obtained by mixing equal volumes of these two fungal agents.

3. The compound microbial agent containing Penicillium citrinum and Trichoderma guizhouense according to claim 2, characterized in that, The liquid culture medium used for the liquid fermentation culture of Penicillium citrinum NJAU-T106 and Trichoderma guizhouense NJAU4742 included PDB liquid medium.

4. The compound fungal agent containing Penicillium citrinum and Trichoderma guizhouense according to claim 2, characterized in that, The conditions for liquid fermentation culture of Penicillium citrinum NJAU-T106 and Trichoderma guizhouense NJAU4742 were: temperature 28-30℃, rotation speed 170-200rpm, and fermentation time 5-7 days.

5. A method for preparing a compound fungal agent containing Penicillium citrinum and Trichoderma guizhouense, characterized in that, The *Penicillium citrinum* is *Penicillium citrinum* (… Penicillium citrinum NJAU-T106, deposited at the China General Microbiological Culture Collection Center (CGMCC) on September 8, 2025, with accession number CGMCC No. 42183; the *Trichoderma guiyuanensis* mentioned is *Trichoderma guiyuanensis* (…). Trichoderma guizhouense NJAU4742 is deposited at the China General Microbiological Culture Collection Center (CGMCC) on April 11, 2016, with accession number CGMCCNo.12166. The preparation of the compound fungal agent containing Penicillium citrinum and Trichoderma guizhouense includes the following steps: Penicillium citrinum NJAU-T106 was inoculated into liquid culture medium for liquid fermentation to obtain a culture broth. The culture broth was filtered to remove mycelia, obtaining Penicillium citrinum NJAU-T106 spore suspension. The Penicillium citrinum NJAU-T106 spore suspension was centrifuged, the supernatant was discarded, the precipitate was resuspended in sterile water, and the spore concentration was adjusted to ≥1×10⁻⁶ with sterile water. 6 1 spore / mL, to obtain Penicillium citrinum NJAU-T106 inoculum; Trichoderma guiyuan NJAU4742 was inoculated into a liquid culture medium for liquid fermentation to obtain a culture broth. The culture broth was filtered to remove mycelia, obtaining Trichoderma guiyuan NJAU4742 spore suspension. The Trichoderma guiyuan NJAU4742 spore suspension was centrifuged, the supernatant was discarded, the precipitate was resuspended in sterile water, and the spore concentration was adjusted to ≥1×10⁻⁶ with sterile water. 6 The inoculum of *Trichoderma guiyuan* NJAU4742 was obtained by spores / mL. The compound fungal agent containing Penicillium citrinum NJAU-T106 and Trichoderma guiyang NJAU4742 was obtained by mixing equal volumes of these two fungal agents.

6. The method for preparing a compound fungal agent containing Penicillium citrinum and Trichoderma guizhouense according to claim 5, characterized in that, The liquid culture medium used for the liquid fermentation culture of Penicillium citrinum NJAU-T106 and Trichoderma guizhouense NJAU4742 included PDB liquid medium.

7. The method for preparing a compound fungal agent containing Penicillium citrinum and Trichoderma guizhouense according to claim 5, characterized in that, The conditions for liquid fermentation culture of Penicillium citrinum NJAU-T106 and Trichoderma guizhouense NJAU4742 were: temperature 28-30℃, rotation speed 170-200rpm, and fermentation time 5-7 days.

8. The application of the compound microbial agent containing Penicillium citrinum and Trichoderma guizhouense as described in any one of claims 1-4 in promoting banana plant growth, increasing banana yield, and preventing banana wilt disease.

9. The application according to claim 8, characterized in that, When applying, the compound microbial agent containing Penicillium citrinum and Trichoderma guizhouense is applied once each during the seedling stage and the vegetative growth stage of banana plants, with 0.8-1L applied to each banana plant each time by root irrigation.

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