Fungus F67 and application

Abstract

The invention belongs to the field of microorganisms, and relates to a fungus F67 and application thereof, the fungus F67 is Lecanicillium saksenae, the fungus F67 is submitted to CGMCC (China General Microbiological Culture Collection Center) on October 9, 2024 and is preserved, and the preservation number is NO.41531. The invention further discloses a preparation method of the fungus F67. The invention provides a fungus F67 capable of effectively constructing a rhizosphere biological barrier system, promoting healthy growth of tobacco plants and providing theoretical support for green prevention and control of tobacco rhizome diseases, and application of the fungus F67.

Description

Technical Field

[0001] The invention belongs to the field of microorganisms and relates to a fungus F67 and an application thereof, and in particular to a fungus separated from tobacco field soil in Yunnan and an application thereof in tobacco growth promotion and black shank disease prevention and control. Background Art

[0002] Tobacco (Nicotiana tabacum L.) is an annual plant of the genus Nicotiana in the Solanaceae family that is not suitable for continuous cropping. Its leaves are round or oval, its petioles are wing-shaped, it blooms in the later period, and its inflorescence is trumpet-shaped or cone-shaped. The flowering and fruiting period is summer and autumn, and the tobacco leaves mature in July and August each year (Yao Jialing and Chen Wei 2017). Tobacco has very important medicinal value. Studies have found that tobacco can reduce swelling and kill viruses, treat back pain and other diseases, and can also produce more than 40 alkaloids (Tian Youqing et al. 2015), some of which have been used for medical treatment. As one of the important economic crops in my country, tobacco plays a vital role in the economic development of society. Due to the restrictions on planting conditions, input cost constraints and economic interests, continuous cropping of tobacco has become a common phenomenon, which in turn causes continuous cropping obstacles and has an adverse effect on tobacco leaf yield and quality (Ling Aifen et al., 2022). Continuous cropping of tobacco will lead to the accumulation of soil-borne pathogens, resulting in frequent soil-borne diseases of economic crops.

[0003] Tobacco black shank is also commonly known as rotten waist and black root. It was first discovered in 1896 and became popular in my country in 1950. Its harm is second only to tobacco virus disease (Shang Zhiqiang 2007; Hu Enchuan et al. 2019). The disease occurs in any growth period of tobacco, and its main manifestations are: the shank turns black, the tobacco leaves turn yellow, drooping when drying in the afternoon, the pith is disc-shaped when cut open, and there are white silks (Zu Qingxue 2022). In the tobacco seedling stage, black spots first appear on the part of the rhizome close to the ground, and then the spots gradually spread to the surrounding areas. In a dry environment, the affected tobacco seedlings will turn brown and eventually die. In contrast, under humid conditions, white mold-like substances, including hyphae and sporangia, will be generated at the lesions. These pathogens can spread rapidly and cause the death of a large number of tobacco seedlings. During the field growth period, water-soaked spots first appear at the base and upper part of the stem, and the base of the stem then turns black and spreads to the upper part. The diseased tobacco leaves will gradually turn yellow from bottom to top, wilt and hang, and usually the whole plant will die within a few days. When the diseased plants are pulled up for observation, the main and secondary roots are found to be rotten; further examination of the shanks of the stems will reveal that the internal tissues have turned black, shrunk into a shape similar to bamboo shoots, and covered with white mold. These symptoms indicate that tobacco has suffered serious threats from diseases during the seedling and field stages, and its spread and development speed is fast, which has a serious impact on 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, 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 the study of plant roots, soil microbiomes with disease-suppressing characteristics prevent pathogens from invading by forming a balanced micro-ecosystem at the roots of plants, the "biological barrier" of the 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 reflect the health of the soil to a certain extent. Large soil microbial biomass, high diversity, and dynamic balance of microbial flora are key factors in the formation of common disease-suppressing soils (Chen Hainian, 2020).

[0006] At present, the prevention and control of black shank in tobacco cultivation mainly relies on chemical agents such as metalaxyl and mancozeb, which has caused the resistance of pathogens to be continuously enhanced and the toxicity to non-target organisms (Yuan Zongsheng et al., 2001), resulting in frequent tobacco root and stem diseases and difficulty in improving tobacco leaf quality. Therefore, screening beneficial microorganisms to prevent and control pathogenic invasion, antagonistic effects on pathogens, and preparing environmentally friendly and efficient biocontrol agents for biological control are of great significance to the green production of tobacco.

[0007] It has been reported that the biocontrol bacteria for the prevention and control of black shank disease are mainly obtained from the rhizosphere of tobacco plants, but there are few studies on rhizosphere fungi. The fungi that have antagonistic effects on tobacco black shank disease mainly include Trichoderma spp., Penicillium spp., Gliocladium spp., etc. Among them, Trichoderma spp. shows a good antagonistic effect on tobacco black shank disease and has been studied the most. Trichoderma can resist the invasion of pathogenic fungi through the lytic enzymes or antimicrobial proteins it secretes, secretes intracellular enzymes to decompose the cell walls of plant pathogenic fungi, such as chitinase, β-glucanase, cellulase and protease, or secretes extracellular enzymes such as glucosidase to degrade toxins produced by pathogens. Most Trichoderma can produce a variety of biologically active substances such as cell wall degrading enzymes and secondary metabolites that have antagonistic effects on plant pathogenic fungi, bacteria and insects, and can also improve the resistance of plants to diseases and pests, promote the growth and development of crops, and increase yield and quality. Therefore, Trichoderma is often used to prevent and control crop diseases and insect pests. It is mixed with fertilizers using certain techniques and methods to make biofertilizer and apply it to the soil to improve soil conditions.

[0008] Studies have shown that wild large fungi often have novel structures and contain unique bioactive compounds. The unique metabolic pathways will promote changes in the microbial population in the soil during the process of biological co-evolution (Jia Mengyuan et al., 2023). At present, there are few biocontrol fungal resources that can prevent and treat tobacco black shank disease, and the disease-inhibiting and growth-promoting functions of existing biocontrol strains are still unclear. Therefore, we screened and used microorganisms isolated from local tobacco field soils in Yunnan to conduct in-dish antagonism tests, identify new biocontrol strains, and use this as a basis to construct a rhizosphere biological barrier system 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 of the invention

[0009] In order to solve the above-mentioned technical problems existing in the background technology, the present invention 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 object, the present invention adopts the following technical solution:

[0011] A fungus F67, characterized in that: the fungus F67 is Lecanicillium saksenae, and the fungus F67 has been submitted to CGMCC for deposit on October 9, 2024, and the deposit number is NO.41531.

[0012] Based on the disease-suppressing and growth-promoting agent formed by the fungus F67 as described above.

[0013] The above-mentioned disease-inhibiting and growth-promoting agents are plant growth-promoting conditioners, plant pathogen inhibitors or biocontrol agents.

[0014] The use of the fungus F67 as described above or the disease inhibitor and growth promoter as described above in preventing and controlling black shank disease.

[0015] The use of the fungus F67 as described above or the disease suppressing and growth promoting agent as described above in preventing and controlling tobacco black shank disease.

[0016] The use of the fungus F67 as described above or the disease suppressing and growth promoting agent as described above to promote the growth and development of tobacco plants.

[0017] The use of the fungus F67 or the disease-suppressing and growth-promoting agent as described above to improve the biological indicators of tobacco plants, wherein the biological indicators include tobacco plant height, tobacco stem thickness, tobacco root length, tobacco maximum leaf width and tobacco maximum leaf length.

[0018] The use of the fungus F67 as described above or the disease-suppressing and growth-promoting agent as described above to promote tobacco photosynthesis, especially to promote the relative content of tobacco chlorophyll, tobacco chlorophyll a content and tobacco chlorophyll b content.

[0019] The use of the fungus F67 as described above or the disease suppressing and growth promoting agent as described above to improve the water utilization of tobacco plants.

[0020] The use of the fungus F67 as described above or the disease suppressing and growth promoting agent as described above to regulate the respiration of tobacco plants.

[0021] The advantages of the present invention are:

[0022] The present invention provides a fungus F67 and its application. The fungus F67 is a fungus isolated from tobacco field soil. The fungus is identified as Lecanococcus saxenae through molecular biological identification. The sterile metabolic liquid of the fungus F67 provided by the present invention has an inhibition rate of 43.61% on tobacco phytophthora, and the growth radius of the pathogen is significantly smaller than that of the control treatment, and can be used for biological control of black shank disease, especially tobacco black shank disease; at the same time, after potted experiments, the fungus F67 provided by the present invention can greatly reduce the incidence of tobacco phytophthora (the incidence is reduced to 20%, and the control effect is 80%); moreover, the fermented bacterial liquid of the fungus F67 provided by the present invention can improve the biological indicators of tobacco plants, promote the growth of tobacco, especially promote the photosynthesis of tobacco, and increase the accumulation of the relative content of chlorophyll in tobacco leaves. In addition, the water use efficiency and intercellular carbon dioxide concentration of the fungus F67 provided by the present invention are comparable to those of CK and PC, reflecting that the antagonistic treatment helps to improve the ability of tobacco plants to use water and regulate the respiration of tobacco plants. The fungus F67 provided by the present invention can significantly increase the relative content of chlorophyll, can optimize the physiological and biochemical characteristics of tobacco, can effectively prevent and control the occurrence of black shank disease, has good biocontrol effect, is environmentally friendly and highly sustainable, and can reduce dependence on chemical agents. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] Figure 1 The colony morphology of the fungus F67 provided by the present invention at different observation angles;

[0024] Figure 2 It is a systematic analysis development tree of the fungus F67 provided by the present invention based on rDNA-ITS sequence;

[0025] Figure 3 The antagonistic ability of the fungus F67 provided by the present invention to tobacco phytophthora (agar block method);

[0026] Figure 4 The inhibitory effect of the metabolites of the fungus F67 provided by the present invention on tobacco phytophthora;

[0027] Figure 5 These are the results of pot test efficacy with different treatments;

[0028] Figure 6 The effect of the fermentation liquid of the fungus F67 provided by the present invention on the relative content of chlorophyll in tobacco leaves;

[0029] Figure 7 The effect of the fermentation liquid of the fungus F67 provided by the present invention on chlorophyll a of tobacco leaves;

[0030] Figure 8 The invention relates to the influence of the fermentation liquid of the fungus F67 on chlorophyll b of tobacco leaves. DETAILED DESCRIPTION

[0031] In order to better explain the present invention, the main contents of the present invention are further illustrated below in conjunction with specific examples, but the contents of the present invention are not limited to the following examples. The technical solutions of the present invention, if not otherwise specified, are conventional techniques in the art, and the reagents or materials, if not otherwise specified, are all derived from commercial channels.

[0032] Example 1: Isolation of fungus F67

[0033] Soil sample collection and soil suspension preparation

[0034] Soil samples were collected from a long-term (8-year) positioning test site (25°42'N, 103°42'E) in Gengde Village, Dapo Township, Zhanyi District, Qujing City, Yunnan Province. The roots of tobacco plants were dug out with soil intact, the above-ground parts were cut off, and the roots with soil were placed in sterile self-sealing bags and transported to the laboratory at 4°C. According to the method of Zhou Yongqiang et al. (2008), the soil attached to the roots of tobacco was shaken off into the self-sealing bag, mixed and weighed 10.0 g, placed in a triangular bottle containing a small amount of quartz sand and 90 ml of sterile water, and shaken on a shaker for 30 minutes before use.

[0035] Isolation, counting, purification and preservation of microorganisms

[0036] The dilution plate smear method was used to spread three different concentrations of soil suspension (10 -3 , 10 -4 , 10 -5 ) was evenly spread on a freshly prepared plate culture medium, the medium used was potato dextrose medium (PDA), and the colony growth was checked after dark culture at 30°C for 4-5 days. The colony number was counted by the plate counting method, and the dominant bacteria with a plate count percentage of >10% were separated and purified to finally obtain fungus F67, which was stored in an ultra-low temperature glycerol tube at -80°C for subsequent experiments.

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

[0038] Morphological characteristics of strains

[0039] The fungus F67 obtained in Example 1 was inoculated into a potato glucose medium (200 g peeled potatoes, 20 g glucose, 20 g agar powder, 1 L tap water), and the colony was observed after culturing in a 30°C constant temperature incubator for 5 days, with reference to the "Handbook of Fungal Identification" and other materials. The colony edge neatness, size, color, shape, transparency and other characteristics were observed and recorded by naked eye, and then potassium hydroxide wet mount examination was performed, and observation was performed under an optical microscope and photographed.

[0040] Depend on Figure 1 (From left to right: front view, back view, and microscope observation (40 times)) It can be seen that fungus F67 grows dense hyphae on PDA medium. The hyphae are circular and spread. The front of the colony is milky white, the back is light yellow, and the spore powder is white. After 3 days of dark culture, it begins to produce rose-red pigment. Under a 40-fold microscope, the hyphae are well developed, and a large number of spores are attached around.

[0041] Molecular biological identification of strains

[0042] Fungal rDNA-ITS sequence analysis was used, and total fungal DNA was extracted using the CTAB method, which was improved based on the methods of He Yueqiu (2000) and Jie et al. (2004). The fungal mycelium (mung bean size) cultured on the PDA plate was collected in a sterile mortar, ground with liquid nitrogen for 5-10 minutes, and transferred to an Eppendorf tube; 600 μL CTAB extract was added, and the mixture was placed in a 65°C water bath for 1 hour, flipped once every 20 minutes; 600 μL Tris saturated phenol: chloroform: isoamyl alcohol (25:24:1) was added, oscillated and mixed, and centrifuged at 6500 rpm for 30 minutes; the supernatant was transferred and repeated the above steps (centrifugation time was 20 minutes); the supernatant was transferred, 1.5 times the volume of anhydrous ethanol was added, gently shaken and mixed, placed at room temperature for 10 minutes, centrifuged at 12000 rpm for 5 minutes, and the supernatant was discarded; the precipitate was washed twice with 70% ethanol, dried at 37°C to form a film; 30 μL TE was dissolved, 0.8% agarose gel electrophoresis was detected (the sample volume was 2.5 μL), and stored at -20°C. Fungal rDNA-ITS sequence PCR amplification used fungal universal primers ITS1 and ITS4. Among them, the sequence of amplification primer ITS1 is: 5'-TCCGTAGGTGAACCTGCGG-3'; the sequence of ITS4 is: 5'-TCCTCCGCTTATTGATATGC-3'. 50.0μL PCR reaction system: 10×buffer5.0μL, dNTP1.0μL, primer ITS1 and primer ITS4 1.0μL each, MgCl23.0μL, 2U / μLTaq enzyme0.5μL, DNA template1.0μL, ddWater37.5μL. Amplification program: 94℃ pre-denaturation 5min; 94℃ denaturation 30s, 52℃ annealing 30s, 72℃ extension 40s, 30 cycles; 72℃ extension 10min. The amplification product was purified and recovered using Tiangen DP204-02 purification kit and sent to Xi'an Qingke Biotechnology Co., Ltd. for sequencing. After proofreading, the obtained sequences were searched by Blast related sequences, homology analysis was performed using ClustalX2.0 software, phylogenetic tree was constructed using Neighbor-Joining method in Mega3.0 software, and the sequences were submitted to Genbank database.

[0043] The strain gene was amplified and sequenced to obtain the sequence of fungus F67, including a 578bp target gene fragment (see sequence list for details). The BLAST search in NCBI was used to compare the sequences included in the database, and strains with detailed information were selected as reference strains. Clustal W multiple alignment was performed, and a phylogenetic evolutionary tree was constructed ( Figure 2The results showed that the similarity between strain F67 and the reference strain Lecanicillium saksenae Ecu 121 was 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, due to its identification, Lecanicillium saksenae F67 was used instead of fungus F67 in subsequent experiments.) was inoculated onto a freshly prepared slant PDA culture medium, and 3 to 5 tubes without contamination and with good colony morphology were selected for preservation. It was deposited in the General Microbiology Center of China Microbiological Culture Collection on October 9, 2024, with the preservation number CGMCC No.41531, and the preservation address is the Institute of Microbiology, Chinese Academy of Sciences, No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing.

[0046] Example 3: Evaluation of the biocontrol effect of Lecanococcus saxena F67 on tobacco black shank disease

[0047] Test strains

[0048] The test strain was the rhizosphere dominant microorganism Lecanococcus saxena F67 which could be cultured in the soil of tobacco fields in Yunnan, and the pathogen was Phytophthoranicotianae which was isolated and purified from the stem base tissue of tobacco plants with black shank disease.

[0049] Bacteria interaction

[0050] The plate confrontation method was used to determine the antibacterial activity of the test strain against the tobacco black shank pathogen. The pathogen was activated on an oat culture medium (100g of oat kernels, 10g of sucrose, 20g of agar powder, and 1L of tap water). A bacterial block (radius 5mm) was punched at the edge of the colony with a hole puncher and inoculated in the center of a new oat culture medium plate. Three antagonistic bacterial blocks (radius 5mm) were symmetrically inoculated around the bacterial block 3cm from the center. Three plates were set up for replication and placed in an incubator. At the same time, an oat culture medium plate inoculated with only pathogenic bacteria cake was set up as a control. The antibacterial results were observed after 5 days. The width of the antibacterial circle was measured by the cross method, and the antibacterial rate was calculated.

[0051] Inhibition rate (%) = (control pathogen colony diameter - treated pathogen colony diameter) / control pathogen colony diameter × 100%.

[0052] Through the indoor plate confrontation test with tobacco phytophthora as the target, it was found that the antagonistic effect of the dominant microorganism in the tobacco rhizosphere, Lecanococcus saxena F67, was better ( Figure 3). The SPSS20 software was used to analyze the antibacterial bandwidth, which was 15.01±0.20 mm, and the antibacterial rate reached 69.56% (Table 1).

[0053] Table 1 The inhibition bandwidth and inhibition rate of Lecanococcus saxenaensis 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, cultured on a shaker at 28°C and 170 r / min, and taken out after 96 hours (the fungus was required to be shaken until the bacterial liquid was viscous and a large number of mycelium groups appeared). The fermentation liquid was centrifuged at 4°C and 10000 r / min for 10 minutes, the supernatant was collected, and filtered with a 0.22 μm filter membrane to obtain the sterile supernatant (cell-free supernatant, CFS) of Saxena lecanii F67.

[0058] The sterile fermentation liquid was mixed with the unsolidified oat culture medium in a ratio of 1:4 and shaken evenly. An equal amount of sterile distilled water and unsolidified oat culture medium were mixed as a control. The mixed liquid 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 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 inhibition rate of the sterile metabolite of the test strain Saxena lecanii F67 on tobacco phytophthora was 43.61%, and the growth radius of the pathogen was significantly smaller than that of the control treatment. In order to further clarify its control effect on black shank disease on tobacco plants, Saxena lecanii F67 was subjected to a tobacco potted bio-verification test.

[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 lecanii F67 on dry shed potted plants

[0064] Materials and Methods

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

[0066] Potted test materials: tobacco coated seeds MS Yunyan 87 (Yuxi China Tobacco Seed Co., Ltd.), V9 seedling medium (Shandong Shouguang Tianfeng Horticultural Materials Factory), fluazifop-acetamiprid (Shaanxi Hengtian Bio-Agriculture Co., Ltd.), 20-20-20 balanced water-soluble fertilizer (Guangdong Weisheng Agriculture Co., Ltd.), 30% carbendazim-methoxam aqueous solution (Henan Bissell Agricultural Technology Co., Ltd.), 200 kg of original soil without human disturbance, seedling trays, transplanting pots, yellow sticky insect boards, and label plates.

[0067] Potted test method: It is divided into four parts: seed raising, tobacco seedling transplanting, test treatment and index determination.

[0068] (1) Seedling cultivation: Carry out in a constant temperature and light incubator, with a temperature of 24.5℃, a humidity of 75%, and a light / darkness of 16h / 8h. Sow 1-2 seeds in each hole to a depth of 0.5-1cm, cover with substrate, preferably barely visible, ventilate for 10 minutes every day and check the growth, and remove weeds in time. For film-covered seedling cultivation, remove the plastic film after 80-90% of the seedlings have emerged, and water with 10ml of tap water every 2 days. Increase the frequency or amount of watering appropriately during the large cross stage (when the fifth true leaf appears), and apply 2-3ml of Hogland nutrient solution to each hole at a fertilizer concentration of 100ppm.

[0069] (2) Transplantation of tobacco seedlings: When the tobacco seedlings have a certain seedling shape at the seedling stage (the eighth true leaf appears), they can be transplanted. Before transplanting, the seedlings should be hardened and no nutrient solution should be used. The tobacco seedlings should be moved to a flower pot with a volume of 5 kg. The soil should be original soil that has not been disturbed by humans. Avoid the surface soil and collect soil with a depth of 10-20 cm. After passing through a 2mm soil sieve, mix it with the substrate in a ratio of 1:1. Carefully transplant the tobacco seedlings that have completed the nursery period into the flower pot with their roots and soil. Compact the soil in the pot by hand to ensure the normal growth of the tobacco seedlings after transplanting. After transplanting, water the roots with 500 mL of water and place them in a scientific research dry shed for growth. Ensure sufficient light and water sources and a stable and high temperature.

[0070] (3) Experimental treatment: After transplanting, the seedlings were stabilized for 21 days, and 200 mL of water was applied every 2 days. The seedlings needed to be hardened before treatment. During the treatment, the spore suspension of tobacco phytophthora and the bacterial solution of Saxena lecanii F67 were poured into the roots to verify the control effect. Ten tobacco plants were treated in each treatment, and the treatment was repeated 3 times. Each treatment was 15 days apart, and normal quantitative topdressing and conventional pest and disease management were carried out during the treatment. The potted treatment settings are shown in Table 3. Each pot was first added with 200 mL of liquid from different treatments, and then 50 mL of pathogen solution was added 2 days later.

[0071] Table 3 Dry shed potted plant test settings

[0072]

[0073] Preparation of tobacco spore suspension: tobacco spore suspension was cultured on OA plates for 21 days, the surface hyphae were scraped, soaked in 0.1% KNO3 solution for 72 hours, and then mixed with sterile water and mashed. The mixture was placed in a 4°C refrigerator for 40 minutes, and then cultured in an incubator at 25°C for 20 minutes. After being taken out, it was prepared into a zoospore suspension (the concentration was determined by dilution and coating to be 9.5×10 6 CFU / mL), add 1% glucose solution for later use. Irrigate the roots of each plant with 50 mL of the prepared concentration of tobacco spore suspension.

[0074] Preparation of biocontrol liquid: Saxena lecanii F67 was activated in PDA liquid medium to obtain seed liquid, which was stored in a 4°C refrigerator. Before each treatment, 10 mL of the liquid was taken from the seed liquid and 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 liquid was taken out and the concentration of the liquid was determined by dilution and coating. The Saxena lecanii F67 liquid was diluted to 10 6 Concentration level: 200 mL of the prepared concentration of Saxena lecanii F67 solution was poured into the roots of each plant.

[0075] 30% carbendazim·oxamyl aqueous solution was dissolved in water at 0.67 mL / L and applied to tobacco roots and soil. Fluazifop·acetamiprid was dissolved in water at 0.27 g / L and sprayed on tobacco leaves. 20-20-20 balanced water-soluble fertilizer was dissolved in water at 0.35 g / L and applied to tobacco roots and soil and sprayed on tobacco leaves regularly according to the growth of tobacco leaves.

[0076] Index determination: After the third treatment, according to "YC / T 142-2010 Tobacco Agronomic Character Survey and Measurement Method", the plant height, stem diameter, root length, number of effective leaves, maximum leaf area, aboveground / underground dry / fresh weight of tobacco plants were measured. Young leaves in the middle of tobacco plants were collected to determine the chlorophyll and photosynthetic indexes of tobacco leaves. The classification of tobacco black shank disease is based on the national industry standard GB / T23222-2008, and the incidence rate, disease index, control effect and relative control effect are statistically calculated. The classification standard (Table 4) and calculation formula are attached.

[0077] Table 4 Grading standards for tobacco black shank disease

[0078]

[0079]

[0080] The calculation formula is:

[0081] (1) Maximum leaf area = leaf area index (0.58) × maximum leaf width × maximum leaf length

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

[0083] (3) Disease index = {∑(number of diseased plants at each level × disease level index)} × 100 / total survey index. (Total survey index = total number of plants or leaves × highest level index)

[0084] (4) Control effect (%) = (incidence rate of control group - incidence rate of treatment group) / incidence rate of control group × 100%.

[0085] (5) Relative efficacy = (F CK -F t ) / F CK ×100%. (F CK represents the disease index of the blank group, F t represents the disease index of the treatment group)

[0086] Potted plant experiment: disease suppression and growth promotion effects

[0087] The control effect of Lecanococcus saxena F67 on tobacco black shank disease: The control effects of the potted test with four treatments are shown in Table 5 and Figure 5 As shown in the figure, compared with the water control group (CK), the pathogen treatment group (PN) had a 0% morbidity rate in CK and a 100% morbidity rate in PN. All tobacco plants infected by the pathogen showed symptoms of black shank disease, indicating that Phytophthora nicotianae had a disease effect on tobacco plants and the symptoms were obvious. Compared with PN, the disease index of the positive control group (PC) and the antagonistic treatment group (F67) was greatly reduced, among which the disease index of F67 dropped from 61.2 to 7.2, the morbidity rate dropped to 20%, the control effect reached 80%, and the relative control effect reached 88%. It shows that strain F67 and traditional chemical control methods have good control effects on tobacco black shank disease.

[0088] Table 5 F67 fermented bacterial liquid pot test results

[0089]

[0090] Effect of Saxena Lecanopsis F67 on the biological traits of tobacco: As shown in Table 6, after exogenous addition of F67 fermentation liquid, the tobacco plant height, stem thickness and root length were the largest and significantly different from those of other treatments, which were 113.67±1.33cm, 12.98±0.53cm and 10.85±1.81cm respectively. The maximum leaf width and maximum leaf length were 16.93±1.05cm and 39.24±1.83cm respectively. The tobacco leaves were stretched and natural in shape, with 18±1 effective leaves and a maximum leaf area of ​​385.38±20.73cm. 2. Although the aboveground / underground fresh weight of F67 was the largest among the four treatments, the difference between the groups was not significant. However, F67 had a certain promoting effect on the accumulation of tobacco dry matter, with its aboveground dry weight reaching 31.50±1.75g ​​and underground dry weight reaching 2.23±0.17g. In summary, F67 fermented bacterial liquid can improve the biological indicators of tobacco plants and promote the growth of tobacco.

[0091] Table 6 Effects of F67 fermentation liquid on biological parameters of tobacco plants

[0092]

[0093] Note: In Table 6, the units of plant height, stem diameter, root length, maximum leaf width and maximum leaf length are all in cm; the units of aboveground fresh weight, underground fresh weight, aboveground dry weight and underground dry weight are all in g; the unit of effective leaf number is piece; the unit of maximum leaf area is cm 2 a, b, and c represent significant differences (arrange the mean values ​​of each group from large to small, mark the letter a after the largest mean, and use this mean to compare with each mean in turn. All insignificant differences are marked with the same letter a until a mean with a significant difference is encountered, and then the letter b is marked. Similarly, the letter c is marked for downward comparisons); ab and bc represent that there are differences between groups but they are not significant.

[0094] Effects of Lecanococcus saxena F67 on tobacco physiology and biochemistry: Results of determination of chlorophyll relative content (SPAD value), chlorophyll a and chlorophyll b under different treatments Figures 6 to 8 As shown. The SPAD values ​​of the water control group (CK), positive control group (PC), antagonistic treatment group (F67), and pathogen 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 fermented bacterial solution was the highest, and there were significant differences among the groups. There was no significant difference in the chlorophyll a content in tobacco leaves among the groups. The measured values ​​of the four treatment groups were all between 0.83 and 0.87 mg / g.FW, but there were significant differences in the content of chlorophyll b. The measured values ​​of the three groups of F67, CK, and PC were between 1.27 and 1.32 mg / g.FW, while the measured value of PN was only 0.80 mg / g.FW. This shows that the exogenous addition of F67 fermented bacterial solution can promote tobacco photosynthesis and increase the accumulation of the relative content of chlorophyll in tobacco leaves.

[0095] The effect of F67 fermentation liquid on the photosynthesis of tobacco plants is shown in Table 7. The net photosynthetic rate of leaves after adding F67 antagonistic treatment reached 8.22±0.60μmol·m -2 ·s -1Compared with the water control group (CK) and the positive control group (PC), the photosynthetic rate of tobacco leaves increased by 68.09% and 41.97%, respectively. There were significant differences among the treatments, indicating that the exogenous addition of F67 fermentation liquid can significantly increase the net photosynthetic rate of tobacco plants. Compared with the other three treatments, F67 significantly increased the transpiration rate and stomatal conductance of tobacco leaves per unit time and per unit area, with the measured values ​​of 2.60±0.27g·m 2 ·h -1 、126.78±27.26mmol·m -2 ·s -1 The water use efficiency and intercellular carbon dioxide concentration of F67 were comparable to those of CK and PC, reflecting that the antagonistic treatment was helpful to improve the ability of tobacco plants to use water and regulate their respiration.

[0096] Table 7 Effect of F67 fermentation liquid on tobacco photosynthesis

[0097]

[0098] Among them, a, b, c, and d represent significant differences (arrange the averages of each group from large to small, mark the letter a after the largest average, and use this average to compare with each average in turn. All insignificant differences are marked with the same letter a until a mean with a significant difference is encountered, and then marked with the letter b. Similarly, compare downwards and mark c and d).

[0099] Due to the infection of pathogens, the net photosynthetic rate, transpiration rate, stomatal conductance and water use efficiency of the pathogen treatment group were much lower than those of the other three treatments, indicating that the plant cells were damaged and could not carry out normal photosynthesis and respiration, resulting in a high intercellular carbon dioxide concentration with significant differences.

[0100] In summary, compared with CK, F67 has a higher relative chlorophyll content and can optimize the physiological and biochemical characteristics of tobacco; compared with PN, it has a better 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 dependence on chemical agents.

Claims

1. A fungus strain F67, characterized in that: The fungus F67 is Lecanicillium saksenae, and the fungus F67 was submitted to CGMCC for deposit on October 9, 2024, and the deposit number is NO.41531.

2. A disease-suppressing and growth-promoting agent formed by the fungus F67 according to claim 1.

3. The disease-inhibiting and growth-promoting agent according to claim 2, characterized in that: The disease-inhibiting and growth-promoting agent is a plant growth-promoting conditioner, a plant pathogen inhibitor or a biocontrol agent.

4. Use of the fungus F67 according to claim 1 or the disease-suppressing and growth-promoting agent according to claim 2 or 3 in preventing and treating black shank disease.

5. Use of the fungus F67 according to claim 1 or the disease-suppressing and growth-promoting agent according to claim 2 or 3 in preventing and controlling tobacco black shank disease.

6. Use of the fungus F67 according to claim 1 or the disease suppressing and growth promoting agent according to claim 2 or 3 in promoting the growth and development of tobacco plants.

7. Use of the fungus F67 according to claim 1 or the disease-suppressing and growth-promoting agent according to claim 2 or 3 in improving biological indicators of tobacco plants, wherein the biological indicators include tobacco plant height, tobacco stem thickness, tobacco root length, tobacco maximum leaf width and tobacco maximum leaf length.

8. Use of the fungus F67 according to claim 1 or the disease-suppressing and growth-promoting agent according to claim 2 or 3 in promoting photosynthesis of tobacco, especially in promoting the relative content of tobacco chlorophyll, the content of tobacco chlorophyll a and the content of tobacco chlorophyll b.

9. Use of the fungus F67 according to claim 1 or the disease suppressing and growth promoting agent according to claim 2 or 3 for improving water utilization of tobacco plants.

10. Use of the fungus F67 according to claim 1 or the disease suppressing and growth promoting agent according to claim 2 or 3 in regulating the respiration of tobacco plants.

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