Fungus f67 and applications

Abstract

The present application belongs to the field of microorganisms, and relates to a fungus F67 and application, wherein the fungus F67 is Lecanicillium saksenae, and the fungus F67 has been preserved in the China General Microbiological Culture Collection Center on October 9, 2024, and the preservation number is CGMCC NO.41531. The present application provides the fungus F67 and application which can effectively construct a rhizosphere biological barrier system, promote the healthy growth of tobacco plants, and provide theoretical support for the green prevention and control of tobacco root and stem diseases.

Description

TECHNICAL FIELD

[0001] The present application belongs to the field of microorganisms, and relates to a fungus F67 and application, in particular to a fungus isolated from tobacco field soil in Yunnan and application in tobacco growth promotion and black shank prevention and control. BACKGROUND

[0002] Tobacco (Nicotiana tabacum L.) is an annual solanaceous plant of the genus Nicotiana, which is not suitable for continuous cropping. The leaves grow in a circular or oval shape, the petiole is winged, and the flowers bloom in the later stage. The inflorescence is trumpet-shaped or conical, and the fruiting period is summer and autumn. The leaves mature in July and August every year (Yao Jialing and Chen Wei 2017). Tobacco has very important medicinal value. Research has found that tobacco can relieve swelling and kill toxins, treat back pain and other diseases, and produce more than 40 alkaloids (Tian Youqing et al. 2015), some of which have been used for medical treatment. As one of China's important economic crops, tobacco plays a crucial role in the economic development of society. Due to the limitations of planting conditions, cost constraints and economic benefits, tobacco continuous cropping has become a common phenomenon, which has caused continuous cropping obstacles and adversely affected tobacco yield and quality (Ling Aifen et al. 2022). Continuous cropping of tobacco can lead to accumulation of soil-borne pathogens, causing frequent occurrence of soil-borne diseases in economic crops.

[0003] Tobacco black shank (Tobacco black shank) is also commonly known as rotten waist and black root. It was first discovered in 1896 and became prevalent in China in 1950, posing a threat second only to tobacco virus disease (Shang Zhiquang 2007; Hu Enchuan et al. 2019). The disease occurs at any growth stage of tobacco and mainly manifests as blackening of the shank, yellowing of the leaves, drooping of the leaves in dry conditions, and disc-shaped and white silk-like appearance of the pith when cut open (Zu Qingxue 2022). In the seedling stage of tobacco, black spots first appear on the part of the rhizome near the ground, and then the disease spreads to the surrounding area. In dry environments, affected seedlings turn brown and eventually die. In contrast, in humid conditions, white mold-like substances, including hyphae and sporangia, are generated at the diseased spots, which can rapidly spread and cause mass mortality of seedlings. In the field growth stage, water-stained lesions first appear on the base and upper part of the stem, and the base of the stem then turns black and spreads upward. Diseased tobacco leaves gradually yellow from bottom to top, wilt and hang down, and usually die within a few days. When the diseased plants are pulled out and observed, it can be seen that the main and auxiliary roots are rotted; further examination of the shank of the stem reveals that the internal tissue is black and shriveled into a thin bamboo-like shape, covered with white mold. These symptoms indicate that tobacco is severely threatened by disease in the seedling and field stages, and the disease spreads and develops rapidly, posing a serious threat to tobacco production (Li Jianlong 2017).

[0004] Tobacco black shank is a soil-borne fungal disease caused by Phytophthora nicotianae. Because the pathogen has strong vitality in the soil and can survive alone in the soil for several months, and some can even survive for several years in very harsh environments, and it is difficult to eliminate in the soil, it has become one of the most serious soil-borne diseases that harm tobacco (Wu Shouming et al., 2023).

[0005] In plant root research, soil microbiomes with disease-suppressive properties prevent pathogen invasion by forming a balanced microecological system within plant roots—a "biological barrier" within the plant roots. Studies have found that the occurrence of soil-borne diseases is closely related to rhizosphere microorganisms (Jiang Jing et al., 2010). Changes in rhizosphere microorganisms can, to a certain extent, reflect the health of the soil. High soil microbial biomass, high diversity, and a dynamic balance of microbial communities are key factors in the development of generally disease-suppressive soils (Chen Hainian, 2020).

[0006] Currently, tobacco cultivation primarily relies on chemical fungicides such as metalaxyl and mancozeb to control black shank. These have led to increasing resistance among pathogens and toxic effects on non-target organisms (Yuan Zongsheng et al., 2001), resulting in frequent root and stem diseases and difficulties in improving tobacco leaf quality. Therefore, screening for beneficial microorganisms that can antagonize pathogens and developing environmentally friendly, highly effective biocontrol agents for biocontrol is of great significance to green tobacco production.

[0007] Reported biocontrol agents for black shank disease are primarily obtained from the rhizosphere of tobacco plants, but research on rhizosphere fungi is limited. Antagonistic fungi against tobacco black shank disease primarily include Trichoderma spp., Penicillium spp., and Gliocladium spp. Among them, Trichoderma spp. exhibits strong antagonistic effects against tobacco black shank disease and has been the most studied. Trichoderma spp. resists invasion by pathogenic fungi through the production of lytic enzymes or antimicrobial proteins. They secrete intracellular enzymes, such as chitinase, β-glucanase, cellulase, and protease, to degrade the cell walls of phytopathogenic fungi, while also secreting extracellular enzymes, such as glucosidases, to degrade toxins produced by pathogens. Most Trichoderma spp. produce a variety of bioactive substances, including cell wall-degrading enzymes and secondary metabolites, that have antagonistic effects on phytopathogenic fungi, bacteria, and insects. These substances can also enhance plant resistance to pests and diseases, promote crop growth and development, and increase yield and quality. Therefore, Trichoderma is often used to prevent and control crop diseases and insect pests. Using certain techniques and methods, it is mixed with fertilizers to make biofertilizer and applied to the soil to improve soil conditions.

[0008] Studies have shown that wild large fungi often have novel structures and unique bioactive compounds, and unique metabolic pathways will promote changes in microbial populations in the habitat soil during biological co-evolution (Jia Mengyuan et al., 2023). At present, the resources of biocontrol fungi with the function of preventing and controlling tobacco black shank disease are less explored, and the disease control and growth promotion functions of the existing biocontrol strains are not clear. Therefore, the microorganisms isolated from the local tobacco field soil in Yunnan are screened for in-vitro antagonistic test to determine new biocontrol strains, and a rhizosphere biological barrier system is constructed based on this to promote the healthy growth of tobacco plants and provide theoretical support for the green prevention and control of tobacco root and stem diseases. SUMMARY

[0009] In order to solve the above technical problems in the background art, the present application provides a fungus F67 and its application, which can effectively construct a rhizosphere biological barrier system, promote the healthy growth of tobacco plants, and provide theoretical support for the green prevention and control of tobacco root and stem diseases.

[0010] In order to achieve the above purpose, the present application adopts the following technical solutions:

[0011] A fungus F67, characterized in that: the fungus F67 is Lecanicillium saksenae, and the fungus F67 has been deposited with the China General Microbiological Culture Collection Center on October 9, 2024, and the deposit number is CGMCC NO. 41531.

[0012] A disease control and growth promotion agent formed based on the fungus F67 as described above.

[0013] The disease control and growth promotion agent described above is a plant growth promoting conditioner, a plant pathogen inhibitor, or a biocontrol preparation.

[0014] Application of the fungus F67 as described above or the disease control and growth promotion agent as described above in the prevention and control of black shank disease.

[0015] Application of the fungus F67 as described above or the disease control and growth promotion agent as described above in the prevention and control of tobacco black shank disease.

[0016] Application of the fungus F67 as described above or the disease control and growth promotion agent as described above in promoting the growth and development of tobacco plants.

[0017] Application of the fungus F67 as described above or the disease control and growth promotion agent as described above in improving the biological indicators of tobacco plants, including tobacco plant height, tobacco stem diameter, tobacco root length, tobacco maximum leaf width, and tobacco maximum leaf length.

[0018] Use of the fungus F67 as described above or the growth-promoting and disease-inhibiting agent as described above in promoting photosynthesis of tobacco, especially in promoting relative content of chlorophyll of tobacco, content of chlorophyll a of tobacco and content of chlorophyll b of tobacco.

[0019] Use of the fungus F67 as described above or the growth-promoting and disease-inhibiting agent as described above in improving water use of tobacco plants.

[0020] Use of the fungus F67 as described above or the growth-promoting and disease-inhibiting agent as described above in regulating respiration of tobacco plants.

[0021] Advantages of the present application are:

[0022] The present application provides a fungus F67 and application, the fungus F67 is isolated from the soil of tobacco field, and is identified by molecular biology, and the fungus is Lecanicillium sasakii.The inhibition rate of the sterile metabolic liquid of the fungus F67 provided by the present application to tobacco blight is 43.61%, and the growth radius of the pathogenic bacteria is obviously smaller than that of the control treatment, so that the fungus F67 can be used for biological control of black shank, especially tobacco black shank;Meanwhile, through the pot experiment, the fungus F67 provided by the present application can greatly reduce the incidence of tobacco blight (the incidence is reduced to 20%, and the control effect reaches 80%);Moreover, the fermentation liquid of the fungus F67 provided by the present application can improve the biological indexes of tobacco plants, promote the growth of tobacco, especially can promote the photosynthesis of tobacco, improve the accumulation of relative content of chlorophyll of tobacco leaf, in addition, the water use efficiency and intercellular carbon dioxide concentration of the fungus F67 provided by the present application are equivalent to CK and PC, which reflects that the antagonistic treatment helps to improve the ability of water use and respiration regulation of tobacco plants.The fungus F67 provided by the present application can significantly improve the relative content of chlorophyll, can optimize the physiological and biochemical characteristics of tobacco, can effectively prevent and control the occurrence of black shank, has good biocontrol effect, is environment-friendly and sustainable, and can reduce the dependence on chemical agents. BRIEF DESCRIPTION OF DRAWINGS

[0023] Figure 1 is the colony morphology of the fungus F67 provided by the present application under different observation angles;

[0024] Figure 2 is the system analysis development tree of the fungus F67 provided by the present application based on rDNA-ITS sequence;

[0025] Figure 3 is the antagonistic ability of the fungus F67 provided by the present application to tobacco blight (agar block method);

[0026] Figure 4 is the inhibition effect of the metabolic product of the fungus F67 provided by the present application on tobacco blight;

[0027] Figure 5is the control effect of different treatment pots on tobacco;

[0028] Figure 6 is the influence of the fermentation bacteria liquid of the fungus F67 provided by the application on the relative content of chlorophyll of tobacco leaves;

[0029] Figure 7 is the influence of the fermentation bacteria liquid of the fungus F67 provided by the application on chlorophyll a of tobacco leaves;

[0030] Figure 8 is the influence of the fermentation bacteria liquid of the fungus F67 provided by the application on chlorophyll b of tobacco leaves. DETAILED DESCRIPTION

[0031] In order to better explain the application, the main content of the application is further illustrated below in combination with specific examples, but the content of the application is not limited to the following examples only. The technical solutions of the application, if not specifically stated, are conventional technologies in the art, and the reagents or materials, if not specifically stated, are from commercial channels.

[0032] Example 1: Isolation of fungus F67

[0033] Soil sample collection and soil suspension preparation

[0034] The soil sample was collected from a long-term (8 years) positioning test site (25°42'N, 103°42'E) in Gengde Village, Dapo Township, Zhenyie District, Qujing City, Yunnan Province, and the root system with soil was dug out, and the aboveground part was cut off. The soil attached to the tobacco roots was shaken into a sterile self-sealing bag according to the method of Zhou Yongqiang et al. (2008), mixed well, and 10.0 g was taken and placed in a triangular flask containing a small amount of quartz sand and 90 ml of sterile water. After shaking on a shaker for 30 min, it was ready for use.

[0035] Microbial separation counting and purification preservation

[0036] Dilution plate smearing method was used to uniformly smear three different concentrations of soil suspension (10 -3 , 10 -4 , 10 -5 ) on freshly prepared plate medium, and the medium used was potato dextrose medium (PDA). After dark culture at 30℃ for 4-5d, the colony growth was observed, the colony number was counted using plate counting method, and the dominant bacteria with plate counting ratio >10% were separated and purified. Finally, the fungus F67 was obtained, which was placed in a -80℃ ultra-low temperature glycerol tube for preservation, and was used for subsequent test.

[0037] Example 2: Identification and preservation of fungus F67

[0038] Strain morphological characteristic identification

[0039] The fungus F67 obtained in Example 1 was inoculated onto potato glucose medium (peeled potato 200 g, glucose 20 g, agar powder 20 g, tap water 1 L) and cultured in a constant temperature incubator at 30°C for 5 days, and the morphology of the colony was observed. The characteristics of the colony edge, such as neatness, size, color, shape, and transparency, were observed with the naked eye and recorded, and then a potassium hydroxide wet film test was performed, and observation was performed using an optical microscope and photographs were taken.

[0040] From the photographs of the fungus F67 obtained in Example 1, it can be seen that the fungus F67 grew dense mycelium on the PDA medium, the mycelium spread in a circular shape, the colony front was milky white, the back was light yellow, the spores were powdery white, and the colony began to produce a rose-colored pigment after 3 days of dark culture. Figure 1 (From left to right: front view, back view, microscope observation (40 times)) It can be seen that the fungus F67 grew dense mycelium on the PDA medium, the mycelium spread in a circular shape, the colony front was milky white, the back was light yellow, the spores were powdery white, and the colony began to produce a rose-colored pigment after 3 days of dark culture. 40 times microscope observation showed that the mycelium developed well and was attached with a large number of spores around.

[0041] Molecular biological identification of the strain

[0042] The total DNA of the fungi was extracted by CTAB method, and the fungi rDNA-ITS sequence analysis was performed according to the methods of He Yueqiu (2000) and Xie Jie et al. (2004). The fungi mycelium (green bean size) cultured on PDA plate was ground in a sterilized mortar for 5-10 min by liquid nitrogen, and then transferred to an Eppendorf tube. 600 μL of CTAB extraction solution was added, and the mixture was shaken and mixed, and then incubated at 65°C for 1 h with shaking every 20 min. 600 μL of Tris-saturated phenol: chloroform: isopropyl alcohol (25:24:1) mixed solution was added, and the mixture was shaken and mixed, and then centrifuged at 6500 rpm for 30 min. The supernatant was transferred, and the above step was repeated (the centrifugation time was 20 min). The supernatant was transferred, 1.5 times the volume of anhydrous ethanol was added, and the mixture was shaken and mixed, and then incubated at room temperature for 10 min, and then centrifuged at 12000 rpm for 5 min, and the supernatant was discarded. The precipitate was washed with 70% ethanol for 2 times, and then dried at 37°C to form a film. 30 μL of TE was added for dissolution, and the mixture was detected by 0.8% agarose gel electrophoresis (the sample amount was 2.5 μL), and then stored at -20°C. The fungi rDNA-ITS sequence PCR amplification was performed by using the fungi universal primers ITS1 and ITS4. The sequence of the amplification primer ITS1 was 5'-TCCGTAGGTGAACCTGCGG-3', and the sequence of the amplification primer ITS4 was 5'-TCCTCCGCTTATTGATATGC-3'. The 50.0 μL PCR reaction system was as follows: 10×buffer 5.0 μL, dNTP 1.0 μL, primer ITS1 and primer ITS4 1.0 μL each, MgCl2 3.0 μL, 2 U / μL Taq enzyme 0.5 μL, DNA template 1.0 μL, and ddWater 37.5 μL. The amplification program was as follows: 94°C pre-denaturation for 5 min; 94°C denaturation for 30 s, 52°C annealing for 30 s, 72°C extension for 40 s, 30 cycles; and 72°C extension for 10 min. The amplification product was purified and recovered by using a DP204-02 type purification kit, and then sent to Xi'an Qikexing Biological Technology Co., Ltd. for sequencing. After the obtained sequence was checked, the Blast related sequence search was performed, the homology analysis was performed by using ClustalX2.0 software, the Neighbor-Joining method in Mega3.0 software was used to construct a phylogenetic tree, and the sequence was submitted to Genbank database.

[0043] The sequence of the fungi F67 was obtained by strain gene amplification and sequencing, including a 578 bp target gene fragment (see the sequence table for details). The sequences in the database were compared by using the BLAST search in NCBI, the strains with detailed information were selected as reference strains, the Clustal W multiple alignment was performed, and a phylogenetic tree was constructed. Figure 2). The results showed that the similarity of strain F67 and reference strain Lecanicillium saksenae Ecu 121 reached 99.65%, and the strains had high homology, so the fungus F67 was identified as Lecanicillium saksenae.

[0044] Strain preservation

[0045] The isolated and purified Lecanicillium saksenae F67 (fungus F67, since it was identified, the following experiments were all replaced by Lecanicillium saksenae F67 instead of fungus F67) was inoculated into freshly prepared slant PDA medium, and 3-5 tubes with good colony morphology were selected and preserved. On October 9, 2024, it was preserved in the General Microbial Culture Collection Center of China Microbial Culture Collection Management Committee, located at No. 3, Yikhina, Beijing Chaoyang District, and the preservation number was CGMCC No. 41531.

[0046] Example 3: Evaluation of the biocontrol effect of Lecanicillium saksenae F67 on tobacco black shank

[0047] Test strain

[0048] The test strain was Lecanicillium saksenae F67, a rhizosphere dominant microorganism in Yunnan tobacco field soil, and the pathogen was Phytophthora nicotianae isolated and purified from the tissue of the stem base of tobacco plants with black shank disease.

[0049] Bacterial interaction

[0050] The plate confrontation method was used to determine the antibacterial activity of the test strain on the pathogen of tobacco black shank disease. The pathogen was activated on oat medium (100 g of oat kernels, 10 g of sucrose, 20 g of agar powder, and 1 L of tap water) plates, and the colony was punched with a puncher at the edge of the colony (radius 5 mm) and inoculated into the center of a new oat medium plate. Three pieces of antagonistic bacteria colony (radius 5 mm) were inoculated symmetrically at a distance of 3 cm from the center, and three plates were set in triplicate. At the same time, oat medium plates inoculated with pathogenic bacteria cakes were set as controls, and the plates were placed in an incubator. After 5 days, the antibacterial results were observed, the cross method was used to measure the width of the antibacterial ring, and the antibacterial rate was calculated.

[0051] Antibacterial rate (%) = (control pathogenic colony diameter - treated pathogenic colony diameter) / control pathogenic colony diameter x 100%.

[0052] Through the indoor plate confrontation test with Phytophthora nicotianae as the target, it was found that the antagonistic effect of Lecanicillium saksenae F67, a tobacco rhizosphere dominant microorganism, was better Figure 3SPSS20 software analysis showed that the inhibition bandwidth was 15.01±0.20 mm, and the inhibition rate reached 69.56% (Table 1).

[0053] Table 1 Inhibitory bandwidth and inhibition rate of Lecanococcus saxena F67

[0054]

[0055] Note: The data in the table are mean ± standard deviation

[0056] Sterile metabolite interactions

[0057] The test strain was inoculated into PDA liquid medium and cultured on a shaker at 28°C and 170 rpm. The culture was removed after 96 hours (the fungus was shaken until the culture liquid became viscous and a large number of mycelial clumps appeared). The fermentation broth was centrifuged at 4°C and 10,000 rpm for 10 minutes, and the supernatant was collected and filtered through a 0.22 μm filter to obtain a sterile supernatant (cell-free supernatant, CFS) of Lecanococcus saxena F67.

[0058] Sterile fermentation broth and unsolidified oat culture medium were mixed and shaken in a ratio of 1:4. An equal amount of sterile distilled water and unsolidified oat culture medium was mixed as a control. The mixture was poured into a plate (90 mm in diameter). After the culture medium solidified, a pathogen cake (2 mm in radius) was inoculated in the center of the plate and cultured at 28°C for 7 days. The diameter of the pathogen colony was measured by the cross-cross method and the inhibition rate of the sterile supernatant of the antagonistic strain was calculated.

[0059] like Figure 4 As shown in Table 2, the sterile metabolite of the test strain, Lecanococcus saxenae F67, inhibited Phytophthora nicotianae by 43.61%, and the growth radius of the pathogen was significantly smaller than that of the control. To further clarify its efficacy against black shank disease in tobacco plants, a bioassay test was conducted using Lecanococcus saxenae F67 in tobacco pots.

[0060] Table 2 Inhibitory activity results of F67 metabolites against tobacco black shank pathogens

[0061]

[0062] Note: The data in the table are mean ± standard deviation

[0063] Example 4: Control Effect of Saxena Lecanopsis F67 on Dry-shed Potted Plants

[0064] Materials and Methods

[0065] Test strains: Lecanococcus saxena F67 and Phytophthora nicotianae.

[0066] Potting test materials: tobacco coated seeds MS Yunyan 87 (Yuxi Zhongyan Seed Co., Ltd.), V9 seedling substrate (Shandong Shouguang Tianfeng Garden Material Factory), fluazinam·nitenpyram (Shaanxi Hengtian Biological Agricultural Co., Ltd.), 20-20-20 balanced water-soluble fertilizer (Guangdong Weisheng Agricultural Co., Ltd.), 30% thiophanate-methyl·hymexazol (Henan Biser Agricultural Technology Co., Ltd.), 200 kg of undisturbed original soil, seedling plug, transplanting flower pot, yellow sticky board, and label card.

[0067] Potting test method: divided into four parts, seedling, transplanting, test treatment and index determination.

[0068] (1) Seedling: in a constant temperature and light incubator, the seedling temperature is 24.5℃, the humidity is 75%, the light / dark is 16h / 8h, 1-2 seeds per hole to 0.5-1cm deep, covered with substrate, and the seedling growth is observed daily for 10 minutes of ventilation and removal of weeds. Covering film seedling is removed after 80-90% of the seedlings emerge, and 10ml of tap water is poured every 2 days. Increase the watering frequency or amount appropriately at the big cross stage (the fifth true leaf appears), and apply 2-3ml of Hoagland's nutrient solution per hole, with a fertilizer concentration of 100ppm.

[0069] (2) Transplanting: transplanting is carried out when the seedlings have a certain plant type at the seedling stage (the eighth true leaf appears). The seedlings are transplanted after hardening off, and no more nutrient solution is used. The seedlings are transplanted into flower pots with a volume of 5kg, and the soil is selected from undisturbed original soil, avoiding the surface soil, and the soil at a depth of 10-20cm is mixed with the substrate at a ratio of 1:1 after passing through a 2mm soil sieve. The seedlings that have completed the seedbed period are carefully transplanted into the flower pots with soil, and the soil in the pots is compacted with the hand to ensure the normal growth of the transplanted seedlings. After transplanting, 500ml of rooting water is poured, and the pots are placed in a research dry shed to ensure sufficient light and water source and stable high temperature.

[0070] (3) Test treatment: after transplanting, the seedlings are stabilized for 21 days, and quantitative watering of 200ml is carried out every 2 days during this period. The seedlings are hardened off before treatment. The treatment is carried out by pouring into the tobacco Phytophthora spore suspension and Sarcocystis neotomata F67 fungus liquid and chemical pesticides to verify the control effect, with 10 tobacco plants per treatment, repeated 3 times, and the interval between each treatment is 15 days. Normal quantitative fertilization and routine disease and pest management are carried out during this period. The potting treatment is set as shown in Table 3, and 200ml of liquid of different treatments is added to each pot first, and 50ml of pathogen liquid is added after 2 days.

[0071] Table 3 Potting test setting in dry shed

[0072]

[0073] Preparation of Phytophthora nicotianae spore suspension: P. nicotianae was cultured on OA plates for 21 days, and the surface mycelium was scraped and soaked in 0.1% KNO3 solution for 72 h. Then, sterile water was added to crush the mixture. The mixture was placed in a refrigerator at 4°C for 40 min, and then cultured in an incubator at 25°C for 20 min. The spore suspension was prepared by dilution and coating, and the concentration was determined to be 9.5 x 10 6 CFU / mL. Then, 1% glucose solution was added, and the spore suspension was ready for use. The spore suspension was used for root irrigation at a concentration of 50 mL per plant.

[0074] Preparation of biocontrol agent liquid: Lecanicillium muscarium F67 was activated in PDA liquid medium to obtain a seed liquid, which was stored in a refrigerator at 4°C. Before each treatment, 10 mL of the seed liquid was inoculated into a new liquid medium, which was placed in a shaker at 30°C and 170 rpm for 5 days. After the culture was completed, the concentration of the liquid was determined by dilution and coating, and the L. muscarium F67 liquid was diluted to a concentration of 10 6 . The L. muscarium F67 liquid was used for root irrigation at a concentration of 200 mL per plant.

[0075] The 30% dimethomorph and fenamidone aqueous solution was applied to the roots and soil of the tobacco plants at a concentration of 0.67 mL / L, the fluazuron and nitenpyram aqueous solution was sprayed on the leaves of the tobacco plants at a concentration of 0.27 g / L, and the 20-20-20 balanced water-soluble fertilizer was applied to the roots and soil of the tobacco plants at a concentration of 0.35 g / L, and was sprayed on the leaves of the tobacco plants according to the growth of the tobacco plants.

[0076] Index determination: After the third treatment, the plant height, stem diameter, root length, number of effective leaves, maximum leaf area, and above / underground dry / fresh weight of the tobacco plants were determined according to the "YC / T 142-2010 Tobacco Agronomic Character Investigation and Measurement Method". The chlorophyll and photosynthetic indexes of the middle tender leaves of the tobacco plants were determined. The disease severity of the tobacco black shank was graded according to the national industry standard GB / T 23222-2008, and the incidence, disease index, control effect, and relative control effect were calculated. The grading standard (Table 4) and calculation formula are as follows.

[0077] Table 4 Tobacco Black Shank Disease Severity Grading Standard

[0078]

[0079]

[0080] The calculation formula is:

[0081] (1) Maximum leaf area = leaf area coefficient (0.58) x maximum leaf width x maximum leaf length

[0082] (2) Incidence (%) = number of diseased plants / total number of plants x 100%.

[0083] (3) Disease index = {∑(each level of diseased plants x the disease level index)} x 100 / total index (total index = total number of plants or total number of leaves x the highest level index)

[0084] (4) Control effect (%) = (disease incidence of the control group - disease incidence of the treatment group) / disease incidence of the control group x 100%.

[0085] (5) Relative control effect = (F CK -F t ) / F CK x 100% (F CK represents the disease index of the blank group, and F t represents the disease index of the treatment group)

[0086] Disease suppression and growth promotion effect of pot experiment

[0087] Preventive and control effects of Lecanicillium muscarium F67 on tobacco black shank: The preventive and control effects of the four treatments in the pot experiment are shown in Table 5 and Figure 5 Fig. 1. Compared with the water control group (CK), the pathogenic treatment group (PN) had a 100% disease incidence, and all the tobacco plants infected with the pathogenic fungus showed symptoms of black shank, indicating that the tobacco Phytophthora had a disease effect on the tobacco plants and the disease symptoms were obvious. Compared with PN, the positive control group (PC) and the antagonistic treatment group (F67) had a significantly reduced disease index, in which the disease index of F67 decreased from 61.2 to 7.2, the disease incidence decreased to 20%, the control effect reached 80%, and the relative control effect reached 88%. This indicates that strain F67 has a good preventive and control effect on tobacco black shank as traditional chemical control methods.

[0088] Table 5 Preventive and control effect results of the pot experiment of F67 fermentation broth

[0089]

[0090] Influence of Lecanicillium muscarium F67 on the biological characteristics of tobacco: As shown in Table 6, after the treatment of exogenous addition of F67 fermentation broth, the plant height, stem diameter, and root length of the tobacco were the largest among the other treatments and had significant differences, being 113.67 ± 1.33 cm, 12.98 ± 0.53 cm, and 10.85 ± 1.81 cm, respectively. The maximum leaf width and maximum leaf length were 16.93 ± 1.05 cm and 39.24 ± 1.83 cm, respectively, the tobacco leaf was stretched and the morphology was natural, the number of effective leaves was 18 ± 1, and the maximum leaf area was 385.38 ± 20.73 cm 2The aboveground and underground fresh weight of F67 was the largest among the four treatments, but the difference between groups was not significant. F67 had a certain promoting effect on the accumulation of dry matter of tobacco, with an aboveground dry weight of 31.50±1.75 g and an underground dry weight of 2.23±0.17 g. In summary, F67 fermentation broth can improve the biological indicators of tobacco plants and promote the growth of tobacco.

[0091] Table 6 Effect of F67 fermentation broth on the biological indicators of tobacco plants

[0092] Note: In Table 6, the units of plant height, stem diameter, root length, maximum leaf width, and maximum leaf length are cm; the units of aboveground fresh weight, underground fresh weight, aboveground dry weight, and underground dry weight are g; the unit of effective leaf number is piece; the unit of maximum leaf area is cm 2 a, b, c represent significant differences (arrange the average numbers from large to small, mark the letter a after the largest average number, compare the average number with each average number in turn, mark the same letter a if the difference is not significant, mark the letter b after the average number that is significantly different, and mark the letter c in the same way) ; ab, bc represent differences between groups but not significant.

[0093] Effect of L. theophylli F67 on the physiology and biochemistry of tobacco: The determination results of relative chlorophyll content (SPAD value), chlorophyll a, and chlorophyll b under different treatments are shown in Table 8. Figure 6 to Figure 8 The SPAD values of the water control group (CK), positive control group (PC), antagonistic treatment group (F67), and pathogenic treatment group (PN) were 43.60±3.60, 45.08±2.63, 49.79±4.04, and 34.70±6.58, respectively. The SPAD value of tobacco leaves treated with F67 fermentation broth was the highest, and there was a significant difference between groups. There was no significant difference in chlorophyll a content in tobacco leaves among the four groups, with measured values between 0.83 and 0.87 mg / g.FW, but there was a significant difference in chlorophyll b content, with measured values between 1.27 and 1.32 mg / g.FW in F67, CK, and PC groups, and only 0.80 mg / g.FW in PN. This indicates that the addition of F67 fermentation broth can promote photosynthesis in tobacco and improve the accumulation of relative chlorophyll content in tobacco leaves.

[0094] Effect of F67 fermentation broth on photosynthesis in tobacco plants, as shown in Table 7. The net photosynthetic rate of leaves treated with F67 antagonistic treatment was 8.22±0.60 μmol·m -2 ·s -1Compared with the clear water control group (CK) and the positive control group (PC), it increased by 68.09% and 41.97% respectively, and there was a significant difference between each treatment, indicating that exogenous addition of F67 fermentation broth could significantly improve the net photosynthetic rate of tobacco plants. F67 significantly improved the transpiration rate and stomatal conductance per unit area per unit time compared with the other three groups, with measured values of 2.60±0.27 g·m 2 ·h -1 、126.78±27.26 mmol·m -2 ·s -1 , and the water use efficiency and intercellular carbon dioxide concentration of F67 were equivalent to those of CK and PC, reflecting that antagonistic treatment helped to improve the water use and respiratory capacity of tobacco plants.

[0095] Table 7 Effect of F67 fermentation broth on tobacco photosynthesis

[0096]

[0097] Among them, a, b, c, d represent significant difference (arrange the average of each group from large to small, mark the letter a after the largest average, compare the average with each average in turn, mark the same letter a if there is no significant difference, until the average with significant difference is encountered, mark the letter b after it, and mark c and d in the same way).

[0098] The net photosynthetic rate, transpiration rate, stomatal conductance, and water use efficiency of the pathogenic treatment group were much lower than those of the other three groups due to the infection of pathogenic bacteria, indicating that the plant cells were damaged and could not perform normal photosynthesis and respiration, resulting in a higher intercellular carbon dioxide concentration and significant difference.

[0099] In summary, compared with CK, F67 has a higher relative content of chlorophyll, which can optimize the physiological and biochemical characteristics of tobacco; compared with PN, it has good biocontrol effect and can effectively prevent and control the occurrence of black shank disease; compared with PC, it is environmentally friendly and sustainable, and can reduce the dependence on chemical agents.

Claims

1. A fungus strain F67, characterized in that: The fungus F67 is Lecanicillium saksenae. The fungus F67 was submitted for deposit to the General Microbiology Center of the China Culture Collection Administration on October 9, 2024, and the deposit number is CGMCC NO.41531.

2. A biocontrol agent comprising the fungus F67 according to claim 1.

3. Use of the fungus F67 according to claim 1 or the biocontrol agent according to claim 2 in inhibiting the activity of tobacco Phytophthora.

4. Use of the fungus F67 according to claim 1 or the biocontrol agent according to claim 2 in controlling tobacco black shank disease, wherein the tobacco black shank disease is caused by Phytophthora nicotianae.

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