Penicillium chrysogenum strain capable of inducing plants to improve disease resistance

Abstract

The invention provides penicillium chrysogenum capable of inducing plants to generate disease resistance, and belongs to the technical field of biocontrol bacteria. The invention provides penicillium chrysogenum. The penicillium chrysogenum and phytophthora nicotianae do not have antagonism. After the spore suspension of the penicillium chrysogenum is sprayed on crops, gene expression can be regulated and controlled from salicylic acid, ethylene and jasmonic acid ways, active oxygen accumulation is reduced, and meanwhile, defensive enzyme activity is improved, so that plants are induced to generate resistance to pathogenic bacteria. The pre-inoculation of penicillium chrysogenum reduces the morbidity of tobacco black shank to a certain extent and slows down the expansion speed of black shank scabs, the treatment of inoculation of penicillium chrysogenum 1 day in advance has a good control effect on tobacco black shank, and the relative control effects are up to 42.86% and 27.08% in 3 days and 7 days.

Description

Technical Field

[0001] This invention belongs to the field of biocontrol bacteria technology, specifically relating to a Penicillium chrysogenum strain that can induce plants to improve their disease resistance. Background Technology

[0002] Phytophthora indicum ( Phytophthora nicotianae Black shank, caused by infection, is one of the major soil-borne diseases of tobacco. Its perennial occurrence and prevalence seriously threaten the safe production of tobacco. Currently, the control of tobacco black shank often combines agricultural and chemical control methods, but these methods are often costly and place significant pressure on the environment's self-regulation. Furthermore, once black shank occurs, it causes irreversible economic losses. Early prevention can reduce the occurrence of black shank to some extent. Most commercially available microbial preparations are preventative, primarily based on bacterial inoculum. These preparations enhance plant resistance to pathogens through antagonism, competition, and induction mechanisms, but currently, the available microbial sources are limited, and the treatment spectrum is singular. Therefore, exploring more usable microbial sources is of great significance for the green production of tobacco.

[0003] In addition to genetically inherited natural barriers, plants have evolved induced defense mechanisms. Induced defense mainly refers to resistance formed after pathogen invasion, primarily involving signal cascade amplification, accumulation of resistance substances, and molecular expression, characterized by its rapid and persistent nature. With the continuous improvement of molecular analysis techniques in recent years, it has been discovered that non-host resistance response (NHR) is built on the same molecular basis as host resistance to pathogen adaptation. In interactions between non-host pathogens and plants, many signals from host resistance participate in non-host defense. Host resistance is mainly triggered by two modes: PTI and ETI. In most cases, PTI and ETI are also the basis of NHR. When plants are exposed to biotic stress, they initiate complex signal transduction mechanisms, coordinating signals to respond to multiple stresses. In plant immunity, reactive oxygen species, antioxidant enzymes, resistance genes, and hormonal changes all lead to changes in plant defense capabilities. Therefore, exploring non-host pathogens is also a way to improve disease resistance. Summary of the Invention

[0004] The present invention provides a Penicillium chrysogenum strain that can induce plants to produce broad-spectrum disease resistance.

[0005] This invention provides a Penicillium chrysogenum strain, which has been biologically preserved with accession number CGMCCNo.42216.

[0006] This invention also provides the application of the above-mentioned Penicillium chrysogenum in inducing plant disease resistance.

[0007] The present invention also provides a fungal agent for inducing plant disease resistance, comprising spores of the aforementioned Penicillium chrysogenum.

[0008] The application further provides a preparation method of the bacterial agent, which comprises culturing the Penicillium chrysogenum in PDA medium for a period of time, collecting mycelium and spores, diluting the spores into a spore suspension with a concentration of >1x10 6 CFU / mL, and adding Tween 20 into the spore suspension and uniformly mixing to obtain the bacterial agent.

[0009] In a preferred mode of the application, the volume percentage of Tween 20 in the bacterial agent is 0.01-0.05%.

[0010] The application further provides application of the bacterial agent or the bacterial agent prepared by the preparation method in improving plant disease resistance.

[0011] In a preferred mode of the application, the plant is tobacco.

[0012] The application further provides a method for inducing a plant to improve disease resistance, which comprises spraying the bacterial agent or the bacterial agent prepared by the preparation method on the plant.

[0013] In a preferred mode of the application, the spraying amount of the bacterial agent is 10-20 mL / plant.

[0014] In a preferred mode of the application, the improvement of disease resistance comprises improvement of disease resistance of the plant during infection of pathogenic fungi; and the pathogenic microorganism comprises Phytophthora nicotianae and Alternaria alternata.

[0015] Beneficial effects: the application provides a Penicillium chrysogenum, which is screened from tobacco stalk extract, and is found to be highly homologous to Penicillium chrysogenum F-94 after ITS amplification and sequencing. The Penicillium chrysogenum in the application does not have antagonistic effect on Phytophthora nicotianae under culture conditions.

[0016] The application takes the resistant variety Yun 87 and the susceptible variety Honghuadajinyuan as materials, inoculates the host pathogenic bacteria Phytophthora nicotianae and the non-host microorganism Penicillium chrysogenum artificially, analyzes the difference in NHR related resistance path after inoculation of the non-host Penicillium chrysogenum and the host pathogenic bacteria Phytophthora nicotianae through biochemical kits, RT-qPCR and ELISA technologies. The results show that after induction of the Penicillium chrysogenum, the resistant variety Yun 87 and the susceptible variety Honghuadajinyuan can induce high expression of related genes in the salicylic acid pathway, the jasmonic acid pathway and the ethylene pathway, increase the defense related enzyme activity, and increase the defense substances such as salicylic acid, jasmonic acid and ethylene. The incidence of tobacco black shank is reduced, the black shank lesion is reduced, and the occurrence and harm of the black shank are effectively prevented.

[0017] Biological preservation information Penicillium chrysogenum YNAU-01 Penicillium chrysogenum , which was preserved in China General Microbiological Culture Collection Center (CGMCC) on September 24, 2025, at No. 1, Xibahe Road, Haidian District, Beijing, China, and the preservation number is CGMCC No. 42216. BRIEF DESCRIPTION OF DRAWINGS

[0018] Figure 1 Fig. 1 is a diagram of morphological observation and molecular biological identification results of Penicillium chrysogenum and Phytophthora nicotianae, wherein present test represents the strains in the examples, and the branch point data represents the bootstrap support rate. Figure 2 Fig. 2 is a diagram of binary culture results of Penicillium chrysogenum and Phytophthora nicotianae, wherein the green colonies on the left represent Penicillium chrysogenum, and the white colonies on the right represent Phytophthora nicotianae. Figure 3 Fig. 3 is a diagram of differences in SA pathway signals under different treatments, wherein * represents a significant difference at the 0.5 level; CK represents a blank control (inoculated with sterile water); Pen represents inoculation with Penicillium chrysogenum; Phy represents inoculation with Phytophthora nicotianae; and HD represents red big; wherein A-B represents expression amount, C-D represents expression amount, E-F represents SA content, G-H represents expression amount, and I represents expression amount. NPR1 WRKY70 Figure 4 Fig. 4 is a diagram of differences in JA pathway signals under different treatments, wherein A-B represents expression amount, C-D represents expression amount, E-F represents expression amount, G-H represents expression amount, and I represents JA content. AOS MYC2 COI1 Figure 5 Fig. 5 is a diagram of differences in ET pathway signals under different treatments, wherein A-B represents expression amount, C-D represents expression amount, E-F represents expression amount, G-H represents expression amount, and I represents ET content. CTR1 EIN3 ERF5 Figure 6 Fig. 6 is a diagram of changes in tobacco black shank disease resistance genes under different treatments, wherein A-B represents expression amount, C-D represents expression amount, E-F represents expression amount, G-H represents expression amount, and I represents expression amount. HSR203J PR-1B Figure 7 Fig. 7 is a diagram of changes in stomata under different treatments, wherein A-B represents photos of stomata, C-D represents stomata opening rate, E-F represents stomata opening degree. Figure 8 Fig. 8 is a diagram of changes in active oxygen content under different treatments, wherein A-B represents ORF content, C-D represents H2O2 content, E-F represents hydroxyl radical content. Figure 9 ​​​​​​​​​​Figure 1 is a diagram of defense enzyme activity changes under the induction of Penicillium chrysogenum, wherein A-B: SOD, C-D: CAT, E-F: POD, G-H: PPO, I-J: PAL; Figure 10 Figure 2 is a diagram of SA pathway signal changes under the induction of Penicillium chrysogenum, wherein A-B: SA content, C-D: NPT content, E-F: NIN content; PR-1B Figure 3 is a diagram of JA pathway signal changes under the induction of Penicillium chrysogenum, wherein A-B: JA content, C-D: OPDA content, E-F: OPGL content; PR-1B Figure 4 is a diagram of active oxygen metabolism changes under the induction of Penicillium chrysogenum, wherein A-B: ORF content, C-D: H2O2 content, E-F: hydroxyl radical content; PR-1B Figure 5 is a diagram of related defense enzyme activity changes after pre-inoculation of Penicillium chrysogenum, wherein A-B: SOD, C-D: CAT, E-F: POD, G-H: PPO, I-J: PAL; Figure 11 Figure 6 is a diagram of black shank occurrence after inoculation of Phytophthora parasitica for 7 days under different treatments. NPR1 WRKY70 Figure 7 is a diagram of black shank occurrence after inoculation of Phytophthora parasitica for 7 days under different treatments. Figure 12 Figure 8 is a diagram of black shank occurrence after inoculation of Phytophthora parasitica for 7 days under different treatments. AOS MYC2 Figure 9 is a diagram of black shank occurrence after inoculation of Phytophthora parasitica for 7 days under different treatments. COI1 Figure 10 is a diagram of black shank occurrence after inoculation of Phytophthora parasitica for 7 days under different treatments. Figure 13 Figure 11 is a diagram of black shank occurrence after inoculation of Phytophthora parasitica for 7 days under different treatments. Figure 14 Figure 12 is a diagram of black shank occurrence after inoculation of Phytophthora parasitica for 7 days under different treatments. Figure 15 DETAILED DESCRIPTION

[0019] The present application provides a Penicillium chrysogenum YNAU-01, and the Penicillium chrysogenum has completed biological preservation, and the preservation number is CGMCC No.42216.

[0020] The Penicillium chrysogenum in the present application is screened from tobacco stalk extract liquid, and ITS sequence sequencing is performed, and the used ITS amplification primers are as follows: ITS1-F (SEQ ID No.1): TCCGTAGGTGAACCTGCGG; ITS4-R (SEQ ID No.2): TCCTCCGCTTATTGATATGC.

[0021] ​​​The present application utilizes the ITS amplification primer for amplification, and the procedure of the amplification comprises: 95 DEG C pre-denaturation for 5 min; then 94 DEG C denaturation for 30 s, 57 DEG C annealing for 30 s, 72 DEG C extension for 90 s, a total of 30 cycles; 72 DEG C extension for 10 min. After amplification, sequencing finds that the sequence of the ITS of the Penicillium chrysogenum in the present application is shown as SEQ ID No. 3.

[0022] The Penicillium chrysogenum in the present application is cultured in a PDA culture medium for sporulation, and spores of the Penicillium chrysogenum are picked up in an LB liquid culture medium and cultured in a 28 DEG C shaker; after 3 days of culture, 200 μL of bacterial liquid is sucked by a pipette and spread on a PDA culture medium, and then placed in a 28 DEG C constant temperature incubator for culture; after 7 days, the Penicillium chrysogenum produces a large number of spores.

[0023] The present application also provides application of the above-mentioned Penicillium chrysogenum in inducing plant resistance to diseases.

[0024] In the examples of the present application, strain confrontation culture is set, and it is found that the Penicillium chrysogenum and the Phytophthora parasitica do not produce an inhibition circle, and even in the later culture stage, the colonies of the Penicillium chrysogenum and the Phytophthora parasitica appear overlapping growth, which proves that the Penicillium chrysogenum does not produce obvious antagonism to the Phytophthora parasitica. However, spore suspension of the Penicillium chrysogenum is sprayed on leaves, and in the salicylic acid pathway, the jasmonic acid pathway, the ethylene pathway and the like, the related genes can be induced to have high expression, the defense related enzyme activity is increased, and the defense substances such as salicylic acid, jasmonic acid and ethylene are increased. The incidence of the Phytophthora parasitica of tobacco is reduced, the Phytophthora parasitica lesion is reduced, and the occurrence and harm of the Phytophthora parasitica are effectively prevented.

[0025] The present application also provides a microbial agent for inducing plant resistance to diseases, comprising spores of the above-mentioned Penicillium chrysogenum.

[0026] The present application also provides a preparation method of the above-mentioned microbial agent, comprising culturing the above-mentioned Penicillium chrysogenum in a PDA culture medium for a period of time, collecting mycelium and spores, diluting the mycelium and spores into a spore suspension with a concentration of not less than 1×10 6 CFU / mL, mixing the spore suspension with Tween 20 to obtain the microbial agent.

[0027] The present application washes the mycelium and spores with sterile water after culturing the Penicillium chrysogenum in a PDA culture medium for 3 days, and filters the mycelium and spores with 4 layers of gauze; then counts the mycelium and spores with a blood cell counting plate, and dilutes the mycelium and spores into a spore suspension with a concentration of about 1×10 6 CFU / mL. 200 μL of Tween 20 is added into 500 mL of the spore suspension to obtain the spore suspension or the microbial agent, and the final volume concentration of Tween 20 in the microbial agent is 0.01-0.05%.

[0028] The application further provides application of the bacterial agent or the bacterial agent prepared by the preparation method in improving plant disease resistance.

[0029] In one embodiment of the application, the bacterial agent is sprayed on the leaves of tobacco to induce high expression of multi-pathway defense substances, such as in the salicylic acid pathway, P. chrysogenum effectively induces tobacco NPR1 gene and WRKY70 gene in the resistant variety K326 and the susceptible variety Honghuadajinyuan, and effectively induces accumulation of salicylic acid in tobacco; in the jasmonic acid pathway, P. chrysogenum effectively induces the resistant variety K326 and the susceptible variety Honghuadajinyuan to AOS and MYC2 up-regulate the expression of gene, and jasmonic acid accumulates more rapidly in the treatment of inoculation with P. chrysogenum, and accumulates at 12 h; in the ethylene pathway, P. chrysogenum effectively induces the resistant variety K326 and the susceptible variety Honghuadajinyuan to CTR1 and ERF5 express gene, and ethylene accumulates to the maximum at 24 h after inoculation; P. chrysogenum can induce high expression of tobacco black shank resistance related gene PR1 , and the expression amount reaches the maximum at 24 h; the occurrence of tobacco black shank is investigated at the 3rd day and the 7th day after inoculation, and the results show that the inoculation with P. chrysogenum has a lower incidence rate and smaller black shank lesion.

[0030] Induction of tobacco resistance-related genes by penicillium chrysogenum and its secondary metabolites: After inducing tobacco resistance by penicillium chrysogenum and its secondary metabolites, the expression of PR-1 gene in the resistant variety Y99 treated by penicillium chrysogenum secondary metabolites was higher than that of other treatments, and the expression was up-regulated by 26.5 times after 3 days; in the susceptible variety HD, the expression of PR-1 gene was lower than that of the untreated, but the reaction was faster, reaching the maximum after 1 day. In the salicylic acid (SA) pathway, the up-regulation of NPR1 and PR4 genes in tobacco treated by penicillium chrysogenum and its secondary metabolites was faster or the expression was higher than that of the untreated, in HD, after inoculation with penicillium chrysogenum secondary metabolites (HPc), the expression of NPR1 gene increased significantly at 1 day, which was 4.1 times of the blank control (HCK) at the same time, and 3.1 times of the pre-inoculation. In Y99, after induction by penicillium chrysogenum secondary metabolites (YPc), the expression of NPR1 gene increased sharply to 15.84 at 2 days after inoculation with tobacco brown spot, which was up-regulated by 12.6 times compared with 0 day, and was up-regulated by 5.0 times and 6.4 times compared with the blank control (YCK) and the treatment of only inoculating tobacco brown spot (YA), respectively. In the jasmonic acid (JA) pathway, in Y99, the expression of PR-6 and COI1 genes in tobacco treated by penicillium chrysogenum and its secondary metabolites was lower than that of the untreated. At the same time, the activities of SOD enzyme and PAL enzyme in tobacco treated by penicillium chrysogenum and its secondary metabolites were higher than those of the rest, in Y99, the activities of SOD enzyme in YPc and YPCN increased first, then decreased, and finally increased again, reaching the maximum at 2 days, which was 1.8 times and 1.3 times of YCK, respectively. It can be seen that penicillium chrysogenum and its secondary metabolites can induce the expression of tobacco defense-related genes in multiple pathways, activate the activity of defense-related enzymes, and enhance the disease resistance of tobacco. Through pot experiment, the disease index of tobacco brown spot was reduced after 1 day of induction of tobacco resistance by penicillium chrysogenum and its secondary metabolites, and the control effect on tobacco brown spot was more than 60% on the 8th day, and more than 34% on the 15th day. Moreover, the size of tobacco brown spot lesion in tobacco induced by penicillium chrysogenum secondary metabolites and penicillium chrysogenum was smaller than that of the rest. The control effect on tobacco brown spot in the field was more than 28%.

[0031] The application also provides a method for inducing plants to improve disease resistance, comprising spraying the above-mentioned fungicide or the fungicide prepared by the above-mentioned preparation method on the plants.

[0032] The spraying amount of the fungicide of the application is 10-20 mL / plant. The improved disease resistance of the application includes improving the disease resistance of plants during the infection process of pathogenic fungi; the pathogenic microorganisms include: tobacco Phytophthora parasitica and tobacco brown spot bacteria.

[0033] In order to further illustrate the present application, a strain of Penicillium chrysogenum for inducing plants to improve disease resistance provided by the present application is described in detail below in combination with examples, but they cannot be understood as limiting the protection scope of the present application.

[0034] In the embodiment of the present application, two tobacco varieties with different resistances are selected, including tobacco black shank resistant variety Yun 87 and susceptible variety Honghuadajinyuan, and the tobacco seeds are provided by Yuxi Zhongyan Seed Company; in the embodiment of the present application, the resistance difference analysis test and the resistance induction test are carried out by using a potting experiment, wherein the floating seedling is carried out in a glass greenhouse of the Plant Protection College of Yunnan Agricultural University, and the potting experiment is carried out in a greenhouse of the Yunnan Diantai Characteristic Agricultural Industrialization Engineering Research Center.

[0035] The resistance difference analysis test: the tobacco seeds are subjected to conventional floating seedling, after germination, the seedlings are thinned, and 20 g of compound fertilizer is added to the seedling pool, the water and fertilizer are changed every 14 days, the roots are scraped and the leaves are cut every 1-2 times a week. When the seedlings are 50 days old, they are transplanted into flowerpots with a diameter of 20.5 cm, a height of 17.5 cm and a bottom diameter of 15 cm. The soil is red soil that has not been planted with tobacco, and 10 g of compound fertilizer (N-P-K=12-8-18) is applied per pot at the time of transplanting. After the seedlings are transplanted and survive, normal water management is carried out. After the tobacco enters the rosette stage, the first topdressing is carried out, 10 g of compound fertilizer per pot, and the second topdressing is carried out after 14 days, topdressing 5 g of urea per pot.

[0036] The resistance induction test: the floating seedling is subjected to seedling, after germination, 20 g of compound fertilizer (N-P-K=15-5-25) is added to the seedling pool, and the roots are scraped and the leaves are cut once at the seedling stage of 30 days, and then once a week. When the seedlings are 50 days old, they are transplanted, and at the time of transplanting, flowerpots with a diameter of 11.7 cm, a height of 9.5 cm and a bottom diameter of 8.5 cm are used, and the soil is red soil that has not been planted with tobacco. Before transplanting, the soil is mixed, and 10 g of compound fertilizer (N-P-K=15-5-25) is applied per pot. After the seedlings survive, normal water management is carried out.

[0037] In the embodiment of the present application, the Penicillium chrysogenum and Phytophthora infestans inoculation test is divided into three treatments, and the complete random test design method is used, and three biological repetitions are carried out. The treatments are as follows: treatment I (CK): sterile water; treatment II (Pen): inoculated with Penicillium chrysogenum; treatment III (Phy): inoculated with Phytophthora infestans; the Penicillium chrysogenum induction inoculation test is divided into four treatments, and the complete random test design method is used, and three biological repetitions are carried out, which are as follows: treatment I (CK): sterile water; treatment II (Phy): inoculated with Phytophthora infestans; treatment III (Pen1): inoculated with Phytophthora infestans after pre-inoculation with Penicillium chrysogenum for 1 day; and treatment IV (Pen3): inoculated with Phytophthora infestans after pre-inoculation with Penicillium chrysogenum for 3 days.

[0038] The data analysis in the embodiments of the present application is performed using Excel for preliminary data analysis, then the average values are compared by software IBMSPSS Statistics 27, the standard deviation is calculated, and Duncan post hoc comparison is used for single factor ANOVA test to analyze the significant differences between treatments. Draw charts using PowerPoint or Origin 2021 software. Microbial data is analyzed by drawing charts on the Gideao cloud platform.

[0039] Example 1 1. Microbial isolation, purification and identification The tobacco Phytophthora was isolated from tobacco plants with typical symptoms of tobacco black shank disease. The tobacco black shank samples were collected from the World Tobacco Variety Garden in Yuxi City, Yunnan Province (24.6497°N, 102.8711°E). The isolation was performed on a clean bench. During isolation, a scalpel was used to cut 5 mm x 5 mm of stem tissue at the junction of the diseased and healthy tissues, which was surface sterilized with 75% alcohol for 2 min, 0.5% sodium hypochlorite solution for 2 min, and sterile water for 4-5 times, each time for 1 min. The sterilized tobacco stem tissue was then placed on PDA medium (20 g glucose, 18 g agar, 200 g potato, 1000 mL water) and incubated in a 28°C incubator. When mycelium grew, the tobacco Phytophthora was isolated and purified, and the purified Phytophthora was transferred to sesame medium (40 g sesame, 18 g agar, 20 g sucrose, 100 mL water) and induced to produce spores under dark conditions at 28°C. The colony morphology of the cultured Phytophthora was observed, and the morphology of the zoosporangium was observed under a general optical microscope for morphological identification.

[0040] The Penicillium chrysogenum was screened from tobacco stem extract. The tobacco stem extract was gradient diluted, and the diluted solution was then applied to PDA medium for coating and incubation in a 28°C incubator. After the growth of microorganisms, the mycelium was picked for purification. After the purified Penicillium strain produced spores, morphological identification was performed based on the spore production structure and conidium morphology of Penicillium under a microscope.

[0041] The morphologically identified Penicillium fungus was scraped from the solid medium, and DNA was extracted. The internal transcribed spacer region was amplified using primers ITS1-F and ITS4-R. The PCR product was detected by 1.5% agarose gel for fragment size, and the PCR product was recovered using a PCR product recovery kit. The recovered PCR product was sent to Shangon Biotech (Shanghai) Co., Ltd. (www.sangon.com) for sequencing. The obtained sequences were compared by NCBI BLAST, and 20 reference sequences highly similar to tobacco Phytophthora and Penicillium, respectively, were downloaded from the NCBI database. A phylogenetic tree based on the neighbor-joining method was constructed using MEGA 11 software.

[0042] Through observation of the morphology of P. nicotianae, the colonies on PDA medium were nearly round, the colony edge was not neat, and the aerial hyphae were developed, showing white cotton-like substance Figure 1 , medium D). Microscopic observation showed that the hyphae were colorless and septate, with special differentiated sporangial stalks, the sporangial stalks were slender and monopodial branching, the sporangia on the top of the sporangial stalks were lemon-shaped, with a papillary protrusion on the top, and no layering phenomenon was observed Figure 1 , medium E-F). After preliminary morphological analysis, it was consistent with the basic characteristics of P. nicotianae.

[0043] The P. chrysogenum colonies were round, with neat edges and white color, and the colonies were greenish after three days of culture, with a large amount of light yellow liquid on the surface, and the colony surface had furrow-like separation Figure 1 , medium A). Microscopic observation showed that a large number of colorless nearly spherical conidia were observed, the conidiophores were colorless, with multiple branches on the top, arranged into a broom shape, the uppermost layer of branches were bottle-shaped, and the top formed a string of conidia Figure 1 , medium B-C), and preliminary analysis was consistent with the basic characteristics of P. chrysogenum.

[0044] The two fungi were identified as P. nicotianae and P. chrysogenum by fungal ITS identification. The phylogenetic tree was constructed by neighbor-joining method, and the isolated P. chrysogenum strain F-94 was in the same branch as P. chrysogenum, with a homology of 99.66%, and the isolated P. nicotianae strain CycC11R was in the same branch as P. nicotianae, with a homology of 100%. It was found by phylogenetic tree construction that P. chrysogenum and P. nicotianae belonged to different genera and had a distant genetic relationship Penicillium chrysogenum Phytophthora nicotianae Figure 1 , medium G) 2. Antagonistic test P. chrysogenum and P. nicotianae were cultured in a binary confrontation, P. chrysogenum and P. nicotianae were cultured on PDA medium, and after 3 days of culture, the colony edge was punched with a 5 mm puncher, and the P. chrysogenum and P. nicotianae cakes were placed at a distance of 2 cm from the edge of the culture dish with tweezers, and then cultured at room temperature (25°C) after inversion, and photographed and recorded at 3 d, 5 d, 10 d, 20 d and 30 d, with 3 replicates, to observe whether there was an inhibition zone.

[0045] P. chrysogenum and P. nicotianae were cultured at room temperature for 30 d, and the results are shown in Figure 2 , within 30 d, P. chrysogenum and P. nicotianae were in contact, no inhibition zone was formed, and at the 30th day, the colonies of the two fungi showed overlapping growth. Therefore, P. chrysogenum and P. nicotianae had no obvious antagonistic effect.

[0046] 3. Spore induction ​​Phytophthora nicotianae was induced to produce sporangia on sesame medium (20 g sucrose, 40 g sesame, 18 g agar, 1000 mL water), and the purified mycelium of P. nicotianae was separated and cultured in LB liquid medium at 28 °C with a shaking speed of 120 rpm / min for 3 days. 200 μL of the bacterial solution was taken with a pipette and evenly spread on the sesame medium, which was then cultured in a 28 °C constant temperature incubator. After 15 days of culture, P. nicotianae formed a large number of zoospores.

[0047] P. nicotianae was induced to produce sporangia on PDA medium, and the spores of P. nicotianae were picked and cultured in LB liquid medium at 28 °C. After 3 days of culture, 200 μL of the bacterial solution was taken with a pipette and evenly spread on the PDA medium, which was then cultured in a 28 °C constant temperature incubator. After 7 days of culture, P. nicotianae produced a large number of spores.

[0048] 4. Inoculation The mycelium and spores of P. nicotianae and P. nicotianae were washed with sterile water. During the washing, about 3 mL of sterile water was added to the culture dish, and the colony surface was scraped several times with a sterile cotton swab. The washing was repeated 2-3 times per dish, and the washing liquid was collected and filtered with 4 layers of gauze. Then, the number of spores was counted with a hemocytometer, and the spore suspension was diluted to contain about 1×10 6 CFU / mL of spores. 200 μL of Tween 20 was added to each 500 mL of spore suspension to complete the preparation of the spore suspension.

[0049] In the resistance difference analysis test, P. nicotianae and P. nicotianae were evenly sprayed on all leaf blades of the tobacco plant.

[0050] In the resistance induction test, after the leaf part was inoculated with P. nicotianae by spraying, P. nicotianae was inoculated on the base of the tobacco stem by wounding inoculation. When inoculating P. nicotianae, the stem surface was first pierced with a 0.35 mm needle, and each tobacco plant was pierced 40 times. Then, about 1 mL of bacterial solution was attached to the pierced part with a cotton swab, and the treatment was kept moist with a plastic wrap.

[0051] 5. Sampling 5.1 Resistance difference analysis sample collection After inoculation with P. nicotianae and P. nicotianae, tobacco leaf samples were collected at 0 h, 12 h, 24 h, 48 h, and 72 h. The collected tobacco leaf samples were quickly stored in liquid nitrogen and then stored in a -80 °C ultra-low temperature freezer in the laboratory for subsequent determination of resistance-related genes, defense enzymes, active oxygen, and metabolites.

[0052] 5.2 Induced resistance research sample collection After inoculation with Phytophthora tobaccoii and Penicillium chrysogenum, tobacco leaf samples were collected at 0 h, 12 h, 24 h, 48 h, 72 h, 96 h, and 168 h, respectively. The collected tobacco leaf samples were quickly placed in liquid nitrogen for preservation and then stored in an ultra-low temperature freezer at -80℃ in the laboratory for subsequent determination of resistance-related genes, reactive oxygen species, and defense enzymes. Stem and leaf samples were collected at 0 d and 7 d for subsequent analysis of the microbial composition of stems and leaves.

[0053] 6. Stomatal observation Stomatal observations were conducted at 0 h, 12 h, 24 h, 48 h, and 72 h after inoculation. Stomatal opening rate and stomatal aperture were measured using gel imprinting-microscopy. Adhesive was evenly applied to the surface of the tobacco leaves, and after 30 minutes of drying, the adhesive was peeled off and observed under a microscope. A 1 cm section was selected for further examination. 2 The dry gels were observed. For each gel, the stomatal aperture of 10 pores and the opening rate of 50 pores were randomly measured. The stomatal opening rate was observed under a 20× field of view. The stomatal opening rate = number of open pores / total number of observed pores. The inner and outer diameters of the pores were measured under a 40× field of view using Imageview software. The stomatal aperture = inner diameter of the pore / outer diameter of the pore.

[0054] There was no significant difference in stomatal opening rate between Yun 87 and *Phytophthora indicum*, but the stomatal aperture of Yun 87 was smaller than that of *Phytophthora indicum*. Both stomatal opening rate and stomatal aperture changed to some extent 12 hours after inoculation with *Phytophthora indicum* and *Penicillium chrysogenum*. Figure 7 (AB). For Yun 87, 12 h after inoculation with Penicillium chrysogenum, both stomatal opening rate and stomatal aperture decreased, with stomatal opening rate decreasing by 5.24% and stomatal aperture decreasing by 1.30%; after inoculation with Phytophthora citrinum, stomatal opening rate decreased by 1.54%, while stomatal aperture increased by 2.13% (AB). Figure 7 (CE). For *Phytophthora safflower*, 12 h after inoculation with *Penicillium chrysogenum*, stomatal opening rate decreased by 3.18% and stomatal aperture decreased by 1.96%. After inoculation with *Phytophthora nicotineae*, stomatal opening rate and stomatal aperture showed the same trend as the control (CK), both showing an increasing trend. Figure 7 (DF). In summary, the stomatal aperture and stomatal opening rate of Penicillium chrysogenum treatment were lower than those of Phytophthora citrinum treatment and control, which is not conducive to the pathogen invading the host through the stomata.

[0055] 7. Determination of relative gene expression levels 7.1 Primer Preparation Based on black shank resistance gene PR-1B (NM_001405376.1) and HSR203J (AB091430.1), salicylic acid pathway gene NPR1 (NM_001326267.1) and WRKY70(NM_001325583.1), jasmonic acid pathway gene AOS (NM_001325375.1), MYC2 (NM_001326072.1) and COI1 (XM_016645934.1), ethylene pathway gene CTR1 (XM_016638635.1), EIN3 (NM_001325921.1) and ERF5 (NM_001326275.1), with Actin (NM_001425880.1) as the internal reference gene, the primers shown in Table 1 were designed, and the primer synthesis was entrusted to Shangon Biotech (Shanghai) Co., Ltd. (www.sangon.com). The synthesized primers were PAGE purified primers.

[0056] Table 1 primer sequence

[0057] 7.2 Sample preparation and expression determination The tobacco leaf samples were ground in a mortar after sterilization with liquid nitrogen. Liquid nitrogen was continuously added during grinding to prevent sample deliquescence. Each sample was ground with liquid nitrogen for 4-5 times until uniform powder without obvious leaf veins was obtained. After RNA was extracted by Trizol method, cDNA was reverse transcribed, and a 20 μl qPCR reaction system was constructed: 2X SYBR Green Pro Taq HS Premix 10 μl, cDNA Template 2 μl (≤100 ng), Primer F (10 μM) 0.4 μl, Primer R (10 μM) 0.4 μl and RNase free water 7.2 μl; Two-step qPCR: 95 °C for 30 s; 95 °C for 5 s, 60 °C for 30 s, 40 cycles. 2 -ΔΔCT The relative expression of the gene was analyzed by the method.

[0058] The gene expression changes of the salicylic acid (SA) pathway are shown in Figure 3 In the resistant variety Yun 87, 0 h to 72 h after inoculation with water, there was no significant change in the expression of NPR1 gene in the control (CK) treatment, and the SA content in the plant leaves was not significantly different. 12 h, 24 h and 48 h after inoculation with Phytophthora parasitica (Phy) and Penicillium chrysogenum (Pen), the expression of NPR1 gene was significantly up-regulated NPR1 in the plant leaves (P < 0.05). Figure 3 A). In the susceptible variety Hongda, 0 h to 72 h after inoculation, there was no significant change in the expression of NPR1 gene in the control (CK) treatmentNPR1 Gene expression levels showed no significant change, and SA content in plant leaves did not differ significantly; Pen and Phy treatments resulted in no significant difference in SA content 12 h after inoculation. NPR1 All genes were significantly upregulated, reaching their maximum expression level at 12 h, and the expression level in the Pen treatment was 2.6 times that in the Phy treatment; 12 h after inoculation, NPR1 Gene expression levels then rapidly decreased. Figure 3 (B). After inoculation with Penicillium chrysogenum and Phytophthora, NPR1 The expression levels of the gene in the Yun 87 variety were higher than those in the Hongda variety, and the peak expression level in the Hongda variety occurred earlier than that in Yun 87. Figure 3 (AB).

[0059] For Yun 87 and Hongda, blank control at 0 h, 12 h, 24 h, 48 h and 72 h after inoculation, NPR1 downstream genes WRKY70 The expression level did not change significantly, and its peak value occurred later than [previous value]. NPR1 Genes. Yun 87, after inoculation with *Phytophthora nicotineae* and *Penicillium chrysogenum*, WRKY70 The expression levels of the gene at all time points were higher than those of the control (CK); and at the same time points, the expression levels of the gene after inoculation with Penicillium chrysogenum (Pen) were significantly higher. WRKY70 Gene expression levels were significantly higher in the Phy treatment than in the Phy treatment. Figure 3 (C). After inoculation with Phytophthora nicotineae and Penicillium chrysogenum, WRKY70 Gene expression levels were all significantly upregulated, and WRKY70 The expression level of the gene was higher in the Pen treatment than in the Phy treatment. Figure 3 (D). After inoculation with Penicillium chrysogenum and Phytophthora, the upregulation multiple of the Yun 87 variety was lower than that of the Hongda variety. After inoculation with Penicillium chrysogenum, WRKY70 The peak expression level of the gene appeared earlier in the susceptible variety Hongda than in Yun 87. After inoculation with the pathogen Phytophthora indicum, the peak expression level appeared earlier in the moderately resistant variety Y87 than in Hongda. Figure 3 Medium CD).

[0060] Comparative analysis of genes related to the jasmonic acid (JA) signaling pathway AOS , MYC2 and COI1 Regarding the expression of *Phytophthora indicum* resistance in the tobacco variety Yun 87, after inoculation with *Phytophthora indicum* and *Penicillium chrysogenum*, AOS All genes showed significant upregulation of expression. Figure 4 (A). For the tobacco variety Red Big, which is susceptible to Phytophthora, Pen and Phy treatments... AOS Gene expression levels were significantly upregulated after inoculation, reaching their maximum at 24 hours. Figure 4 (Middle B). Yun 87 and Hongda after inoculation with Penicillium chrysogenum and Phytophthora citrinum. AOSThe gene change trends were consistent, showing an initial upregulation followed by a downregulation. At 24 hours after inoculation with Phytophthora infestans, the upregulation fold of the Yun 87 variety was higher than that of the Hong Da variety. After inoculation with Penicillium chrysogenum... AOS The upward adjustment factor for Cloud 87 is also higher than that for Red 87.

[0061] Genes related to the jasmonic acid signaling pathway were analyzed after inoculation with Phytophthora nicotina and Penicillium chrysogenum at 0 h, 12 h, 24 h, 48 h, and 72 h. MYC2 Regarding the expression of the resistant variety Yun 87, all treatments MYC2 All genes showed significant upregulation of expression. Figure 4 (C). For the susceptible variety Red Big, all treatments... MYC2 Gene expression levels were significantly upregulated after inoculation. Figure 4 (D). Yun 87 and Hongda after inoculation with Penicillium chrysogenum and Phytophthora citrinum. MYC2 The gene expression trends were consistent, both showing an initial upregulation followed by a downregulation. After inoculation with Phytophthora infestans, the upregulation fold of Yun 87 was lower than that of Hong Da; similarly, after inoculation with Penicillium chrysogenum, the upregulation fold of Yun 87 was also lower than that of Hong Da. Figure 4 Medium CD).

[0062] Genes related to the jasmonic acid signaling pathway were analyzed after inoculation with Phytophthora nicotina and Penicillium chrysogenum at 0 h, 12 h, 24 h, 48 h, and 72 h. COI1 Regarding the expression of information, for cloud 87, the inoculation treatment Phy and Pen COI1 Gene expression was significantly upregulated, with Pen treatment showing the highest upregulation of 1.6-fold and Phy treatment the highest upregulation of 2.2-fold. Phy treatment showed the highest upregulated expression level, significantly higher than the CK treatment. Figure 4 (E). For red, Pen and Phy are processed. COI1 Gene expression levels were significantly upregulated after inoculation. Figure 4 (F). Yun 87 and Hongda after inoculation with Penicillium chrysogenum and Phytophthora citrinum. COI1 The gene change trends were consistent, all showing an upward trend. After inoculation with Phytophthora infestans, the upregulation fold of Yun 87 was lower than that of Hong Da; after inoculation with Penicillium chrysogenum, the upregulation fold of Yun 87 was also lower than that of Hong Da. Figure 4 Medium EF).

[0063] Comparative analysis of genes related to the ethylene (ET) signaling pathway after inoculating *Penicillium chrysogenum* and *Phytophthora nicotine* into *Tobacco Cloud 87* and *Safflower Dajinyuan* at 0 h, 12 h, 24 h, 48 h, and 72 h was performed. CTR1 , EIN3 and ERF5 The expression status. For the resistant variety Yun 87, 24 hours after inoculation with Phytophthora citrinum and Penicillium chrysogenum, CTR1 All genes showed upregulated expression, with the Pen treatment showing a significantly higher fold increase than the CK and Phy treatments.Figure 5 Fig. 6. Expression of CTR1 in resistant tobacco variety Yun 87 and susceptible tobacco variety Hongda after inoculation with P. cinnabarinus and P. parasitica. Fig. 7. Expression of CTR1 in resistant tobacco variety Yun 87 and susceptible tobacco variety Hongda after inoculation with P. cinnabarinus and P. parasitica. CTR1 The expression of CTR1 was up-regulated after 24 h of inoculation, and the up-regulation fold of Pen treatment was higher than that of Phy treatment. The expression of CTR1 in Pen and Phy treatments was significantly higher than that in CK treatment. Figure 5 Fig. 6. Expression of CTR1 in resistant tobacco variety Yun 87 and susceptible tobacco variety Hongda after inoculation with P. cinnabarinus and P. parasitica. Fig. 7. Expression of CTR1 in resistant tobacco variety Yun 87 and susceptible tobacco variety Hongda after inoculation with P. cinnabarinus and P. parasitica. CTR1 The expression of CTR1 was up-regulated after 24 h of inoculation, and the up-regulation fold of Pen treatment was higher than that of Phy treatment. The expression of CTR1 in Pen and Phy treatments was significantly higher than that in CK treatment. CTR1 Fig. 6. Expression of CTR1 in resistant tobacco variety Yun 87 and susceptible tobacco variety Hongda after inoculation with P. cinnabarinus and P. parasitica. Fig. 7. Expression of CTR1 in resistant tobacco variety Yun 87 and susceptible tobacco variety Hongda after inoculation with P. cinnabarinus and P. parasitica. CTR1 The expression of CTR1 was up-regulated after 24 h of inoculation, and the up-regulation fold of Pen treatment was higher than that of Phy treatment. The expression of CTR1 in Pen and Phy treatments was significantly higher than that in CK treatment. Figure 5 Fig. 6. Expression of CTR1 in resistant tobacco variety Yun 87 and susceptible tobacco variety Hongda after inoculation with P. cinnabarinus and P. parasitica. Fig. 7. Expression of CTR1 in resistant tobacco variety Yun 87 and susceptible tobacco variety Hongda after inoculation with P. cinnabarinus and P. parasitica.

[0064] Fig. 6. Expression of CTR1 in resistant tobacco variety Yun 87 and susceptible tobacco variety Hongda after inoculation with P. cinnabarinus and P. parasitica. Fig. 7. Expression of CTR1 in resistant tobacco variety Yun 87 and susceptible tobacco variety Hongda after inoculation with P. cinnabarinus and P. parasitica. EIN3 The expression of CTR1 was up-regulated after 24 h of inoculation, and the up-regulation fold of Pen treatment was higher than that of Phy treatment. The expression of CTR1 in Pen and Phy treatments was significantly higher than that in CK treatment. EIN3 Fig. 6. Expression of CTR1 in resistant tobacco variety Yun 87 and susceptible tobacco variety Hongda after inoculation with P. cinnabarinus and P. parasitica. Fig. 7. Expression of CTR1 in resistant tobacco variety Yun 87 and susceptible tobacco variety Hongda after inoculation with P. cinnabarinus and P. parasitica. Figure 5 The expression of CTR1 was up-regulated after 24 h of inoculation, and the up-regulation fold of Pen treatment was higher than that of Phy treatment. The expression of CTR1 in Pen and Phy treatments was significantly higher than that in CK treatment. EIN3 Fig. 6. Expression of CTR1 in resistant tobacco variety Yun 87 and susceptible tobacco variety Hongda after inoculation with P. cinnabarinus and P. parasitica. Fig. 7. Expression of CTR1 in resistant tobacco variety Yun 87 and susceptible tobacco variety Hongda after inoculation with P. cinnabarinus and P. parasitica. EIN3 The expression of CTR1 was up-regulated after 24 h of inoculation, and the up-regulation fold of Pen treatment was higher than that of Phy treatment. The expression of CTR1 in Pen and Phy treatments was significantly higher than that in CK treatment. Figure 5 Fig. 6. Expression of CTR1 in resistant tobacco variety Yun 87 and susceptible tobacco variety Hongda after inoculation with P. cinnabarinus and P. parasitica. Fig. 7. Expression of CTR1 in resistant tobacco variety Yun 87 and susceptible tobacco variety Hongda after inoculation with P. cinnabarinus and P. parasitica. EIN3 The expression of CTR1 was up-regulated after 24 h of inoculation, and the up-regulation fold of Pen treatment was higher than that of Phy treatment. The expression of CTR1 in Pen and Phy treatments was significantly higher than that in CK treatment. EIN3 Fig. 6. Expression of CTR1 in resistant tobacco variety Yun 87 and susceptible tobacco variety Hongda after inoculation with P. cinnabarinus and P. parasitica. Fig. 7. Expression of CTR1 in resistant tobacco variety Yun 87 and susceptible tobacco variety Hongda after inoculation with P. cinnabarinus and P. parasitica. Figure 5 Fig. 6. Expression of CTR1 in resistant tobacco variety Yun 87 and susceptible tobacco variety Hongda after inoculation with P. cinnabarinus and P. parasitica. Fig. 7. Expression of CTR1 in resistant tobacco variety Yun 87 and susceptible tobacco variety Hongda after inoculation with P. cinnabarinus and P. parasitica.

[0065] Fig. 6. Expression of CTR1 in resistant tobacco variety Yun 87 and susceptible tobacco variety Hongda after inoculation with P. cinnabarinus and P. parasitica. Fig. 7. Expression of CTR1 in resistant tobacco variety Yun 87 and susceptible tobacco variety Hongda after inoculation with P. cinnabarinus and P. parasitica. ERF5 The expression of CTR1 was up-regulated after 24 h of inoculation, and the up-regulation fold of Pen treatment was higher than that of Phy treatment. The expression of CTR1 in Pen and Phy treatments was significantly higher than that in CK treatment. ERF5 Fig. 6. Expression of CTR1 in resistant tobacco variety Yun 87 and susceptible tobacco variety Hongda after inoculation with P. cinnabarinus and P. parasitica. Fig. 7. Expression of CTR1 in resistant tobacco variety Yun 87 and susceptible tobacco variety Hongda after inoculation with P. cinnabarinus and P. parasitica. Figure 5(E). For the susceptible variety Red Big, Pen and Phy treatments. ERF5 Gene expression was upregulated 12 hours after inoculation, reaching its highest level at 48 hours. The upregulation fold in Pen treatment was higher than that in Phy treatment. The upregulation levels in Pen and Phy treatments were significantly higher after inoculation. ERF5 The expression levels were significantly higher than those in the CK treatment. Figure 5 (F). Comparative analysis of resistant and susceptible varieties Yun 87 and Hongda after inoculation with Penicillium chrysogenum and Phytophthora citrinum. ERF5 Genetic variation trends were analyzed, and the results showed that the two varieties exhibited a consistent trend, showing a single peak within 72 hours of inoculation. However, after inoculation with Phytophthora infestans, the upregulation fold of Yun 87 was higher than that of Hong Da, and after inoculation with Penicillium chrysogenum... ERF5 The multiplier for Yun87 is also higher than that for Hongda ( ). Figure 5 Medium EF).

[0066] After inoculating *Penicillium chrysogenum* and *Phytophthora nicotinica* onto tobacco cloud 87 and *Safflower Dajinyuan* at 0 h, 12 h, 24 h, 48 h, and 72 h, the resistance genes for tobacco black shank were compared and analyzed. HSR203J The changes. After inoculation with Phytophthora indica. HSR203J Gene expression was upregulated, with the upregulation fold in Hongda being higher than that in Yun87, and significantly higher than that in CK and Pen treatments. Figure 6 (AB).

[0067] Comparative analysis of black shank resistance genes in two tobacco varieties at 0 h, 12 h, 24 h, 48 h, and 72 h after inoculation with Phytophthora nicotinae and Penicillium chrysogenum. PR-1B The changes were observed in Yun 87 after inoculation with Phytophthora nicotine and Penicillium chrysogenum. PR-1B All genes showed upregulated expression, with Pen treatment showing a more rapid upregulation than Phy treatment, and a higher peak expression level, significantly higher than CK and Phy treatments. Figure 6 (C). For Red Giant, both Pen and Phy treatments showed some upregulation of PR-1B gene expression after inoculation, with the upregulation fold in Pen treatment being higher than that in Phy treatment. Both Pen and Phy treatments showed [further details regarding post-inoculation upregulation]. PR-1B The expression levels were significantly higher than those in the CK treatment. Figure 6 (D). Yun 87 and Hongda after inoculation with Penicillium chrysogenum and Phytophthora citrinum. PR-1B The gene expression trends were consistent, showing an initial upregulation followed by a downregulation. After inoculation with *Phytophthora infestans* and *Penicillium chrysogenum*, the fold increase in PR-1B gene expression in the Yun 87 variety was higher than that in the Hongda variety. Figure 6 Medium CD).

[0068] Penicillium chrysogenum was inoculated into Tobacco Cloud 87 and Red Flower Dajinyuan 1 day and 3 days in advance, followed by Phytophthora nicotineae. The expression of the tobacco black shank resistance gene PR-1B was compared and analyzed at 0 h, 12 h, 24 h, 48 h, and 72 h after inoculation. The results are as follows:Figure 10 As shown in FIG. 6, for the resistant variety Yun 87, the relative expression of PR-1B gene was significantly up-regulated in both inoculation with Phy and induction with Pen. Compared with 0 h, the highest up-regulation of CK treatment was 7.8 times at 12 h, the highest up-regulation of Phy treatment was 12.3 times at 24 h, the highest up-regulation of Pen1 treatment was 38.1 times at 72 h, and the highest up-regulation of Pen3 treatment was 37.6 times at 24 h. The up-regulation of Pen1 treatment was the highest, and the Phy treatment and Pen3 treatment showed a trend of first up-regulation and then down-regulation. For Hongda, the expression was up-regulated in each treatment, and the PR-1B gene was significantly up-regulated after inoculation in Phy, Pen1 and Pen3 treatments, and the highest expression of each treatment was higher than that of the control. The relative expression of PR-1B gene in Yun 87 and Hongda showed the same trend, and the relative expression of PR-1B gene in Yun 87 was higher than that in Hongda. The gene expression was up-regulated in both varieties after inoculation with Phy, and the up-regulation in Yun 87 was higher than that in Hongda. Figure 10 ).

[0069] The expression of salicylic acid signal pathway related genes at 0 h, 12 h, 24 h, 48 h and 72 h after inoculation was compared and analyzed. As shown in FIG. 7, for Yun 87, the relative expression of all treatments was up-regulated after inoculation, and the highest expression of Pen1 treatment was the highest (FIG. 7A). For Hongda, the relative expression of all treatments was up-regulated after inoculation, and the highest expression of Pen1 treatment was the highest (FIG. 7B). The expression of each treatment showed a trend of first up-regulation and then down-regulation. The expression of PR-1B gene was up-regulated in both varieties after inoculation with Phy, and the up-regulation in Yun 87 was slightly higher than that in Hongda. After pre-inoculation with Pen, the up-regulation in Hongda was higher than that in Yun 87. NPR1 WRKY70 The expression of salicylic acid signal pathway related genes at 0 h, 12 h, 24 h, 48 h and 72 h after inoculation was compared and analyzed. As shown in FIG. 7, for Yun 87, the relative expression of all treatments was up-regulated after inoculation, and the highest expression of Pen1 treatment was the highest (FIG. 7A). For Hongda, the relative expression of all treatments was up-regulated after inoculation, and the highest expression of Pen1 treatment was the highest (FIG. 7B). The expression of each treatment showed a trend of first up-regulation and then down-regulation. The expression of PR-1B gene was up-regulated in both varieties after inoculation with Phy, and the up-regulation in Yun 87 was slightly higher than that in Hongda. After pre-inoculation with Pen, the up-regulation in Hongda was higher than that in Yun 87. Figure 11 NPR1 NPR1 Figure 11 NPR1 Figure 11 NPR1

[0070] The expression of salicylic acid signal pathway related genes at 0 h, 12 h, 24 h, 48 h and 72 h after inoculation was compared and analyzed. As shown in FIG. 7, for Yun 87, the relative expression of all treatments was up-regulated after inoculation, and the highest expression of Pen1 treatment was the highest (FIG. 7A). For Hongda, the relative expression of all treatments was up-regulated after inoculation, and the highest expression of Pen1 treatment was the highest (FIG. 7B). The expression of each treatment showed a trend of first up-regulation and then down-regulation. The expression of PR-1B gene was up-regulated in both varieties after inoculation with Phy, and the up-regulation in Yun 87 was slightly higher than that in Hongda. After pre-inoculation with Pen, the up-regulation in Hongda was higher than that in Yun 87. WRKY70 WRKY70 Figure 11 ​​​​​​​​​​The relative expression of the genes was up-regulated in all treatments after inoculation, and the up-regulation was the highest in the Phy treatment. The relative expression of the genes was up-regulated first and then down-regulated in the Phy, Pen1 and Pen3 treatments, and the up-regulation was the highest in the Pen1 treatment. Figure 11 The relative expression of the genes was up-regulated in all treatments after inoculation, and the up-regulation was the highest in the Phy treatment. The relative expression of the genes was up-regulated first and then down-regulated in the Phy, Pen1 and Pen3 treatments, and the up-regulation was the highest in the Pen1 treatment.

[0071] The relative expression of the genes was up-regulated in all treatments after inoculation, and the up-regulation was the highest in the Phy treatment. The relative expression of the genes was up-regulated first and then down-regulated in the Phy, Pen1 and Pen3 treatments, and the up-regulation was the highest in the Pen1 treatment. AOS MYC2 COI1 The relative expression of the genes was up-regulated in all treatments after inoculation, and the up-regulation was the highest in the Phy treatment. The relative expression of the genes was up-regulated first and then down-regulated in the Phy, Pen1 and Pen3 treatments, and the up-regulation was the highest in the Pen1 treatment. Figure 12 The relative expression of the genes was up-regulated in all treatments after inoculation, and the up-regulation was the highest in the Phy treatment. The relative expression of the genes was up-regulated first and then down-regulated in the Phy, Pen1 and Pen3 treatments, and the up-regulation was the highest in the Pen1 treatment. AOS The relative expression of the genes was up-regulated in all treatments after inoculation, and the up-regulation was the highest in the Phy treatment. The relative expression of the genes was up-regulated first and then down-regulated in the Phy, Pen1 and Pen3 treatments, and the up-regulation was the highest in the Pen1 treatment. Figure 12 The relative expression of the genes was up-regulated in all treatments after inoculation, and the up-regulation was the highest in the Phy treatment. The relative expression of the genes was up-regulated first and then down-regulated in the Phy, Pen1 and Pen3 treatments, and the up-regulation was the highest in the Pen1 treatment. Figure 12 The relative expression of the genes was up-regulated in all treatments after inoculation, and the up-regulation was the highest in the Phy treatment. The relative expression of the genes was up-regulated first and then down-regulated in the Phy, Pen1 and Pen3 treatments, and the up-regulation was the highest in the Pen1 treatment. AOS The relative expression of the genes was up-regulated in all treatments after inoculation, and the up-regulation was the highest in the Phy treatment. The relative expression of the genes was up-regulated first and then down-regulated in the Phy, Pen1 and Pen3 treatments, and the up-regulation was the highest in the Pen1 treatment. AOS The relative expression of the genes was up-regulated in all treatments after inoculation, and the up-regulation was the highest in the Phy treatment. The relative expression of the genes was up-regulated first and then down-regulated in the Phy, Pen1 and Pen3 treatments, and the up-regulation was the highest in the Pen1 treatment.

[0072] The relative expression of the genes was up-regulated in all treatments after inoculation, and the up-regulation was the highest in the Phy treatment. The relative expression of the genes was up-regulated first and then down-regulated in the Phy, Pen1 and Pen3 treatments, and the up-regulation was the highest in the Pen1 treatment. MYC2 The relative expression of the genes was up-regulated in all treatments after inoculation, and the up-regulation was the highest in the Phy treatment. The relative expression of the genes was up-regulated first and then down-regulated in the Phy, Pen1 and Pen3 treatments, and the up-regulation was the highest in the Pen1 treatment. MYC2 The relative expression of the genes was up-regulated in all treatments after inoculation, and the up-regulation was the highest in the Phy treatment. The relative expression of the genes was up-regulated first and then down-regulated in the Phy, Pen1 and Pen3 treatments, and the up-regulation was the highest in the Pen1 treatment. MYC2 The relative expression of the genes was up-regulated in all treatments after inoculation, and the up-regulation was the highest in the Phy treatment. The relative expression of the genes was up-regulated first and then down-regulated in the Phy, Pen1 and Pen3 treatments, and the up-regulation was the highest in the Pen1 treatment. Figure 12 The relative expression of the genes was up-regulated in all treatments after inoculation, and the up-regulation was the highest in the Phy treatment. The relative expression of the genes was up-regulated first and then down-regulated in the Phy, Pen1 and Pen3 treatments, and the up-regulation was the highest in the Pen1 treatment. Figure 12 The relative expression of the genes was up-regulated in all treatments after inoculation, and the up-regulation was the highest in the Phy treatment. The relative expression of the genes was up-regulated first and then down-regulated in the Phy, Pen1 and Pen3 treatments, and the up-regulation was the highest in the Pen1 treatment.

[0073] The relative expression of the genes was up-regulated in all treatments after inoculation, and the up-regulation was the highest in the Phy treatment. The relative expression of the genes was up-regulated first and then down-regulated in the Phy, Pen1 and Pen3 treatments, and the up-regulation was the highest in the Pen1 treatment. COI1 The relative expression of the genes was up-regulated in all treatments after inoculation, and the up-regulation was the highest in the Phy treatment. The relative expression of the genes was up-regulated first and then down-regulated in the Phy, Pen1 and Pen3 treatments, and the up-regulation was the highest in the Pen1 treatment. COI1 ​​The expression of all genes was up-regulated, and Pen1 treatment reached the highest peak at 72 h, with the highest up-regulation. Figure 12 For Hongda, all treatments were up-regulated after inoculation, and the Phy treatment had the highest up-regulation fold (Fig. 4C). Figure 12 For the two varieties, the relative expression of the gene in Yun 87 was higher than that in Hongda before inoculation (Fig. 4F). COI1 After inoculation with P. parasitica, the relative expression of the gene in Hongda was higher than that in Yun 87 (Fig. 4F). COI1 The up-regulation fold of the gene in Hongda was higher than that in Yun 87.

[0074] 8. Determination of plant hormone content (Elisa) At 0 h, 12 h, 24 h, 48 h, and 72 h after inoculation, the contents of SA, JA, and ET in the tobacco leaves were determined by enzyme-linked immunoassay. The determination was performed using an Elisa kit from Jiangsu Meimian Industrial Co., Ltd., and was strictly in accordance with the instructions.

[0075] The accumulation of SA at 0 h, 12 h, 24 h, 48 h, and 72 h after inoculation with P. chrysogenum and P. parasitica in two different varieties was compared, and the results are shown in Figs. 4E and F. Figure 3 After inoculation with P. chrysogenum, the SA content in the tobacco plants continued to increase, reaching a maximum at 72 h, and the SA content in Hongda was slightly higher than that in Yun 87. After inoculation with P. parasitica, the SA content reached a maximum at 24 h, and then decreased. The SA content accumulated more and lasted longer in the Phy treatment.

[0076] The accumulation of JA at 0 h, 12 h, 24 h, 48 h, and 72 h after inoculation with P. chrysogenum and P. parasitica in two different varieties was compared. After inoculation with P. chrysogenum, the jasmonic acid content in Yun 87 and Hongda accumulated at 12 h, but the Phy treatment accumulated less at 24 h, and then accumulated to a certain extent (Figs. 4G and H). Figure 4

[0077] The accumulation of ET at 0 h, 12 h, 24 h, 48 h, and 72 h after inoculation with P. chrysogenum and P. parasitica in two different varieties was compared. After inoculation with P. chrysogenum and P. parasitica, the change in ethylene content in the leaves was different from that in the CK treatment. The CK treatment first decreased and then increased, while the Phy and Pen treatments accumulated after 12 h of inoculation, and no obvious accumulation was observed in the control (Figs. 4G and H). Figure 5 ​​

[0078] 8. Active oxygen content determination Determination of superoxide anion (OFR), hydrogen peroxide (H2O2) and hydroxyl radical (OH - ) three active oxygen. Using superoxide anion kit, hydrogen peroxide content kit, hydroxyl radical kit for determination (G0116W, G0112W, G0153W, Suzhou Gairen Biological Technology Co., Ltd.). The determination method refers to its instruction manual. All indicators are determined by spectrophotometry, and determined by enzyme marker instrument (ReadMax 1900, Shanghai Shanshu Biological Technology Co., Ltd.).

[0079] OFR content change: Yun 87 and Honghuadajinyuan showed similar trend, Pen and Phy treatment significantly increased OFR accumulation after inoculation, Pen treatment superoxide anion content accumulation was significantly higher than CK and Phy treatment. Compared with two varieties, susceptible variety Hongda Phy and Pen treatment OFR accumulation was significantly higher than Yun 87 (P Figure 8 A-B).

[0080] H2O2 change: Yun 87 and Honghuadajinyuan showed similar trend, Pen and Phy treatment significantly increased H2O2 accumulation after inoculation. Yun 87 Pen treatment H2O2 accumulation was higher than Phy treatment, Hongda Pen treatment H2O2 accumulation was lower than Phy treatment (P Figure 8 C-D).

[0081] OH - change: Yun 87 and Honghuadajinyuan Pen and Phy treatment showed similar trend after inoculation, Pen and Phy treatment significantly increased OH - accumulation after inoculation, which were significantly higher than CK treatment, Phy treatment OH - accumulation peak was higher than Pen treatment (P Figure 8 E-F).

[0082] Penicillium chrysogenum was inoculated into tobacco Yun 87 and Honghuadajinyuan 1 d and 3 d in advance, and then inoculated with Phytophthora parasitica, and the accumulation of key active oxygen OFR, H2O2 and OH - was compared and analyzed. The results are shown in Figure 13 : for Yun 87, OFR of all treatments was up-regulated, compared with the control, OFR accumulation of each treatment increased 12 h after inoculation with Phytophthora, and the highest OFR accumulation of other treatments was higher than that of the control. Phy treatment OFR content showed an upward trend, Pen1 treatment OFR content increased and then decreased, Pen treatment increased after inoculation with Penicillium chrysogenum, and then decreased, and its content increased again after inoculation with Phytophthora (P Figure 13(A). For Red Giant, OFR content increased in all treatments after inoculation with Penicillium chrysogenum and Phytophthora citrinum, with all treatments showing upregulation of OFR content. Compared with the control, the Phy and Pen1 treatments had higher accumulation levels. Figure 13 (B). Compared to the two varieties, the OFR content in Yun 87 was lower than that in Hong Da. After inoculation with Phytophthora infestans, the OFR accumulation in Hong Da was higher. However, the increase in OFR in Yun 87 after inoculation with Penicillium chrysogenum was higher than that in Hong Da. Figure 13 (AB).

[0083] The accumulation of key reactive oxygen species (H2O2) was compared and analyzed at 0 h, 12 h, 24 h, 48 h, and 72 h post-inoculation. For Yun 87, the H2O2 content increased significantly in all treatments, with the Phy treatment showing the highest accumulation. Compared with the control, the H2O2 accumulation in all treatments after inoculation with Phytophthora was higher, and accumulation began as early as 12 h. The Phy and Pen1 treatments showed the same trend. Figure 13 (C) For Red Giant, after inoculation with Penicillium chrysogenum and Phytophthora citrinum, the accumulation of H2O2 in each treatment showed a trend of first increasing and then decreasing. H2O2 accumulation was observed in each treatment at 12 h, and the accumulation in each treatment was higher than that in the CK treatment (C). Figure 13 (D). In uninoculated Yun 87 and Hongda, the accumulation of H2O2 was not significantly different. After inoculation with Phytophthora citrinum, the increase in hydrogen peroxide in Hongda was higher than that in Yun 87. After inoculation with Penicillium chrysogenum, the increase in hydrogen peroxide in Hongda was also higher than that in Yun 87.

[0084] Comparative analysis of key reactive oxygen species (OH) at 0 h, 12 h, 24 h, 48 h and 72 h post-inoculation. - The accumulation of hydroxyl radicals was observed. For Yun 87, the content of hydroxyl radicals was increased after inoculation with *Penicillium chrysogenum* and *Phytophthora nicotineae*. All treatments showed an increase in hydroxyl radical content at 12 h, with the Phy treatment showing the largest increase. The CK, Phy, and Pen1 treatments showed a trend of first increasing and then decreasing. Figure 13 (E). For *Phytophthora chinensis*, OH- content accumulated in all treatments after inoculation with *Phytophthora nicotine* and *Penicillium chrysogenum*. Compared with 0 h, accumulation occurred in all treatments except Phy at 12 h, but the Phy treatment showed the highest accumulation at 96 h. Figure 13 (F). OH after inoculation with *Safflower 'Big Golden Yuan'*. - The increase was smaller than that of Yun87, but Yun87 had already shown a decline at 96 h, while Honghua Dajinyuan still showed an increasing trend.

[0085] 9. Assay for defensive enzyme activity The activities of superoxide dismutase (SOD), peroxidase (POD), polyphenol oxidase (PPO), phenylalanine ammonia-lyase (PAL) and catalase (CAT) were determined using superoxide dismutase kit, catalase kit, peroxidase kit, polyphenol oxidase kit, phenylalanine ammonia-lyase kit (G0101W, G0105W, G0107W, G0113W, G0114W, Suzhou Genesee Bio-technology Co., Ltd.), and the determination method was according to the instruction manual. All indexes were determined by spectrophotometry, and determined by enzyme marker instrument (ReadMax 1900, Shanghai Shenshu Bio-technology Co., Ltd.).

[0086] CK treatment had no significant difference in SOD enzyme activity in Yun 87 and Hongda, and the SOD enzyme activity was up-regulated at 12 h after inoculation of Penicillium chrysogenum and Phytophthora parasitica. In Yun 87, the SOD enzyme activity was the highest after Pen treatment for 24 h, and the highest after Phy treatment for 48 h (Fig. 1A). Figure 9 In Hongda, the SOD enzyme activity was the highest after Pen treatment for 48 h, and the highest after Phy treatment for 24 h (Fig. 1B). Figure 9 In Hongda, the SOD enzyme activity was the highest after Pen treatment for 48 h, and the highest after Phy treatment for 24 h (Fig. 1B).

[0087] After inoculation of Phytophthora parasitica and Penicillium chrysogenum, the CAT activity was significantly up-regulated (Fig. 2A). Figure 9 For Hongda, the up-regulation fold of Pen treatment was higher than that of Phy treatment (Fig. 2D). Figure 9 For Hongda, the up-regulation fold of Pen treatment was higher than that of Phy treatment (Fig. 2D).

[0088] Compared with the control, the POD enzyme activity was up-regulated after inoculation of Phytophthora parasitica and Penicillium chrysogenum (Fig. 3A). Figure 9 For Hongda, the POD enzyme activity was the highest after Pen treatment for 48 h, and the highest after Phy treatment for 48 h (Fig. 3F). Figure 9 For Hongda, the POD enzyme activity was the highest after Pen treatment for 48 h, and the highest after Phy treatment for 48 h (Fig. 3F).

[0089] The PPO enzyme activity was up-regulated after inoculation with P. chrysogenum and P. parasitica. The Pen and Phy treatments in Yun 87 had the highest up-regulation at 24 h and 48 h, and the Pen and Phy treatments in Hongda had the highest up-regulation at 24 h and 48 h. The up-regulation of the Pen treatment was higher than that of the Phy treatment, and the highest up-regulation of the Pen treatment occurred earlier than that of the Phy treatment. The PPO activity of all treatments in the two varieties was up-regulated to varying degrees, and the up-regulation of the P. chrysogenum and P. parasitica treatments was significantly higher than that of the CK treatment. The PPO activity of the two varieties increased first and then decreased after inoculation Figure 9 I-J).

[0090] For Yun 87, the PAL activity was the highest after 24 h of inoculation with the Pen treatment, and the PAL activity was the highest after 24 h of inoculation with the Phy treatment. The PAL activity of the Pen treatment increased significantly more than that of the Phy treatment Figure 9 G). For Hongda, the PAL activity of the Pen and Phy treatments was the highest after 24 h of inoculation, and the PAL activity of the Pen treatment increased significantly more than that of the Phy treatment Figure 9 H). The PAL activity of Yun 87 was higher than that of Hongda at 0 h of inoculation. After inoculation with P. chrysogenum and P. parasitica, the PAL activity was up-regulated, and the up-regulation of the Pen treatment was higher than that of the Phy treatment. Compared with the control, the PAL activity of the Pen and Phy treatments was up-regulated after inoculation, and both showed an increasing trend first and then a decreasing trend.

[0091] P. chrysogenum was inoculated into Yun 87 and Hongda 1 d and 3 d in advance, and then P. parasitica was inoculated. The changes in the activities of the tobacco resistance-related defense enzymes (SOD, POD, CAT, PPO, and PAL) at 0 h, 12 h, 24 h, 48 h, and 72 h after inoculation were compared and analyzed. The results are shown in Figure 14 For Yun 87, the SOD activity of each treatment was up-regulated at 12 h, and the highest SOD activity occurred in the Pen1 treatment. The Phy treatment had the highest up-regulation. The Phy treatment and the Pen1 treatment showed similar trends Figure 14 A). For HD, the SOD activity of each treatment was up-regulated, and the Pen1 treatment had the highest peak activity and the highest up-regulation Figure 14 B). The enzyme activity of Yun 87 was higher than that of Hongda after inoculation with P. parasitica, and the response speed of the enzyme activity was faster than that of Hongda at 12 h of inoculation.

[0092] The changes of POD activity of tobacco resistance related defense enzymes at 0 h, 12 h, 24 h, 48 h and 72 h after inoculation were compared and analyzed. For Yun 87, the activity of each treatment was up-regulated at 12 h after inoculation with tobacco Phytophthora and C. ochracea, showing a gradual upward trend, and the POD enzyme activity of each treatment was higher than that of CK. Among them, Pen1 treatment was up-regulated the highest, and the up-regulation of each treatment was higher than that of CK Figure 14 in medium C). For Hongda, each treatment was up-regulated after inoculation with tobacco Phytophthora and C. ochracea. The up-regulation of each treatment was higher than that of CK Figure 14 in medium D). After inoculation with tobacco Phytophthora, the up-regulation of POD enzyme activity of Yun 87 was higher than that of Hongda, and at 12 h, the response activity of Yun 87 POD was also higher than that of Hongda, and showed a rising trend.

[0093] The changes of CAT activity of tobacco resistance related defense enzymes at 0 h, 12 h, 24 h, 48 h and 72 h after inoculation were compared and analyzed. For Yun 87, the CAT activity of each treatment was up-regulated at 12 h, and the up-regulation of the two treatments inoculated with C. ochracea in advance was significantly higher than that of other treatments Figure 14 in medium E). For Hongda, the CAT activity of each treatment was up-regulated after inoculation, and the highest CAT activity of each treatment was higher than that of CK. The peak value of Phy treatment was the highest, and the up-regulation of the treatment inoculated with C. ochracea in advance was significantly higher than that of the other treatments at 12 h Figure 14 in medium F). Before inoculation with Phytophthora, the CAT activity of Yun 87 was higher than that of Hongda, and after inoculation with tobacco Phytophthora, the up-regulation of Hongda CAT activity was higher than that of Yun 87.

[0094] The changes of PPO activity of tobacco resistance related defense enzymes at 0 h, 12 h, 24 h, 48 h and 72 h after inoculation were compared and analyzed. For Yun 87, each treatment had an up-regulation trend after inoculation, among which Pen3 treatment was up-regulated the highest, and the PPO activity of each treatment had a trend of first increasing and then decreasing Figure 14 in medium G). For Hongda, each treatment had a certain up-regulation Figure 14 in medium H). After inoculation with tobacco Phytophthora, the up-regulation of PPO activity of Yun 87 was higher than that of Hongda, and the PPO response activity was higher at 12 h.

[0095] The changes of PAL activity of tobacco resistance related defense enzymes at 0 h, 12 h, 24 h, 48 h and 72 h after inoculation were compared and analyzed. For Yun 87, the PAL activity of each treatment increased after inoculation, and the activity peak was higher than that of CK Figure 14 in medium I). For Hongda, each treatment showed a trend of first increasing and then decreasing, and the peak value of Pen1 treatment was the highest. The up-regulation of each treatment was higher than that of the control, and the up-regulation of Pen3 treatment was the highest Figure 14PAL enzyme activity of Yun 87 was higher than that of Hongda, and the up-regulation of PAL enzyme activity of Hongda was higher than that of Yun 87 after inoculation of P. parasitica. The PAL enzyme activity of both varieties increased at 12 h, and showed the trend of first increasing and then decreasing.

[0096] 10. Investigation of black shank disease The incidence of black shank disease was investigated at 3 d and 7 d after inoculation of P. parasitica. The investigation was carried out according to the standard of GB-T23222-2008, and 30 plants were investigated for each treatment, and 15 plants for each variety. Then, the size of stem lesion of black shank disease was measured, and 5 plants were measured for each variety. The incidence and disease index were calculated according to the formula: Incidence = Incidence of diseased plants / Total number of plants × 100%, Disease index = 100 × ∑(disease grade × incidence of diseased plants) / (total number of investigated plants × highest grade), Relative control efficiency (%) = (disease index of control area - disease index of treatment area) / disease index of control area × 100.

[0097] The incidence of black shank disease was investigated after pre-inoculation of P. chrysogenum and non-pre-inoculation of P. chrysogenum. The results showed that no black shank disease was observed in the CK treatment with sterile water, and a high incidence of black shank disease was observed at 3 d in the treatment with P. parasitica. Compared with the treatment with P. parasitica, the incidence of black shank disease was reduced in the treatments with pre-inoculation of P. chrysogenum for 3 d and 7 d. For the resistant variety Yun 87, compared with the treatment with P. parasitica, the incidence of black shank disease was reduced by 13.34% in the treatments with pre-inoculation of P. chrysogenum for 1 d and 3 d at 3 d. The disease index was reduced by 7.41 in the Pen1 treatment and by 5.19 in the Pen3 treatment. At the same time, the size of lesion of black shank disease was significantly reduced in the treatments with pre-inoculation of P. chrysogenum. Figure 15The relative control effect of Penl treatment was higher than that of Pen3 treatment at 3d and 7d.

[0098] The difference of the occurrence of black shank between the resistant tobacco variety Yun 87 and the susceptible tobacco variety Hongda was compared, and the results showed that neither Yun 87 nor Hongda showed disease after being inoculated with P. nicotianae. The disease incidence and disease index of Hongda were higher than those of Yun 87 at 3d and 7d after being inoculated with P. nicotianae. The relative control effect of Yun 87 was higher at 3d after being inoculated with P. nicotianae. The difference between the relative control effects of the two varieties gradually decreased at 7d after being inoculated with P. nicotianae, and the relative control effect of Yun 87 was slightly higher than that of Hongda.

[0099] Table 2 Occurrence of black shank of tobacco under different treatments

[0100] Although the above examples make a detailed description of the present application, it is only a part of the embodiments of the present application, but not all the embodiments. Other embodiments can be obtained according to the present embodiments without creativity, and these embodiments all belong to the protection scope of the present application.

Claims

1. A strain of Penicillium chrysogenum, characterized in that, The said Penicillium chrysogenum has been biologically preserved with the preservation number of CGMCC No. 42216.

2. The use of the Penicillium chrysogenum in claim 1 in inducing plant resistance to diseases.

3. A microbial agent for inducing disease resistance in plants, characterized by, Spores of the Penicillium chrysogenum in claim 1.

4. The method for preparing the bacterial agent of claim 3, characterized by, The preparation of the microbial inoculum includes culturing the Penicillium chrysogenum of claim 1 in PDA medium for a period of time, collecting mycelium and spores, diluting to a spore suspension of >1 x 10 6 CFU / mL, adding Tween 20 to the spore suspension, and mixing to homogeneity to obtain the microbial inoculum.

5. The preparation method according to claim 4, characterized in that, The volume percentage of Tween 20 in the said bacterial agent is 0.01-0.05%.

6. The use of the bacterial agent in claim 3 or the bacterial agent prepared by the preparation method in claim 4 or 5 in improving plant resistance to diseases.

7. Use according to claim 6, characterized in that, The said plant is tobacco.

8. A method of inducing enhanced disease resistance in a plant, comprising, The bacterial agent in claim 3 or the bacterial agent prepared by the preparation method in claim 4 or 5 is sprayed on the plant.

9. The method of claim 8, wherein, The spraying amount of the bacterial agent is 10-20 mL per plant.

10. The method of claim 8, wherein, The said improvement of resistance to diseases includes the improvement of plant resistance to diseases during the infection of pathogenic fungi; the said pathogenic microorganisms include: Phytophthora nicotianae and Alternaria alternata.

Images