Bacillus amyloliquefaciens JQ-4 for antagonizing pathogenic bacteria of tobacco target leaf spot and application of bacillus amyloliquefaciens JQ-4

Abstract

The invention discloses bacillus amyloliquefaciens JQ-4 for antagonizing pathogenic bacteria of tobacco target leaf spot and application of the bacillus amyloliquefaciens JQ-4, and belongs to the technical field of screening of antagonistic strains of the pathogenic bacteria of the tobacco target leaf spot, bacillus amyloliquefaciens is JQ-4 and is named as bacillus amyloliquefaciens J in taxonomy, and bacillus amyloliquefaciens JQ-4 is named as bacillus amyloliquefaciens JQ-4 in taxonomy and is named as bacillus amyloliquefaciens JQ-4 in taxonomy. The strain is applied to preparation of a microbial preparation for preventing and treating tobacco target leaf spot pathogenic bacteria, the prevention effect is improved, and the pathogenic bacteria are prevented from generating drug resistance.

Description

Technical Field

[0001] The invention relates to the technical field of screening strains antagonistic to tobacco target spot pathogens, and more specifically to Bacillus amyloliquefaciens JQ-4 antagonistic to tobacco target spot pathogens and applications thereof. Background Art

[0002] Tobacco target spot is a fungal disease caused by Rhizoctonia solani. After infection with Rhizoctonia solani, tobacco leaves are perforated and torn, seriously affecting tobacco yield and quality. Tobacco target spot was first discovered in Liaoning, my country, and then reported in Jilin, Guangxi, Yunnan, Hunan and Sichuan. In recent years, it has shown a trend of expansion and the damage has become more serious year by year. It has gradually become the main disease of tobacco, seriously affecting the economic benefits of tobacco cultivation.

[0003] The hyphae of the pathogen of tobacco target spot can survive in the soil for many years. The main method for tobacco target spot is to prevent its occurrence and then combine it with treatment to achieve the effect of prevention and control. The prevention and control methods of tobacco target spot mainly include chemical, agricultural and biological control. Among them, chemical control is widely used and practical, but it brings more and more prominent problems such as pesticide residues and pathogen resistance. Agricultural control is time-consuming, labor-intensive and cumbersome. Therefore, low-cost, residue-free, non-toxic and efficient biological control is one of the hot spots in the current research on the prevention and control of tobacco target spot.

[0004] At present, the related biocontrol bacteria used for biological control are mostly Pseudomonas, Streptomyces, Bacillus, etc. Among them, Bacillus bacteria are extremely abundant in the rhizosphere of plants. They have fast reproduction speed, high biosafety, broad-spectrum disease resistance and growth-promoting effects. They are gradually becoming a research hotspot for biocontrol bacteria and are gradually moving towards production and application. Bacillus biocontrol bacteria have great application prospects in the prevention and control of tobacco target spot disease.

[0005] Therefore, how to provide a Bacillus amyloliquefaciens that can antagonize tobacco target spot pathogens is a technical problem that needs to be solved urgently by those skilled in the art. Summary of the invention

[0006] In view of this, the present invention screened out a Bacillus amyloliquefaciens JQ-4 that antagonizes the tobacco target spot pathogen. The strain can effectively antagonize the tobacco target spot pathogen. The strain is applied to the prevention and control of tobacco target spot, which can effectively inhibit the proliferation of the pathogen and provide a theoretical basis for the biological control of tobacco target spot.

[0007] In order to achieve the above object, the present invention adopts the following technical solution:

[0008] A Bacillus amyloliquefaciens JQ-4 antagonistic to tobacco target spot pathogens, characterized in that the Bacillus amyloliquefaciens JQ-4 was deposited in the China Center for Type Culture Collection on December 13, 2023, with a deposit number of CCTCC NO: M 20232552, a deposit address of Wuhan University, Wuhan, China, and a taxonomic name of Bacillus amyloliquefaciens JQ-4.

[0009] As an inventive concept identical to the above technical solution, the present invention also seeks to protect the use of the sterile fermentation liquid of Bacillus amyloliquefaciens JQ-4 in the preparation of a microbial preparation antagonistic to the pathogen of tobacco target spot disease.

[0010] As an inventive concept identical to the above technical solution, the present invention also seeks to protect a microbial preparation for antagonizing tobacco target spot pathogens, wherein the microbial preparation comprises a sterile fermentation broth of Bacillus amyloliquefaciens JQ-4.

[0011] As the same inventive concept as the above technical solution, the present invention also requests the use of the Bacillus amyloliquefaciens JQ-4 in the preparation of preparations antagonistic to tobacco brown spot pathogen, tobacco black shank pathogen, pepper phytophthora, and pepper white rot pathogen.

[0012] Through the above technical scheme, it can be known that compared with the prior art, the present invention screened a strain of Bacillus amyloliquefaciens that can antagonize tobacco target spot disease pathogens from tobacco fields with severe tobacco target spot disease in Hunan tobacco areas. Through the plate confrontation experiment, it was found that the antibacterial rate of the strain against tobacco target spot disease was as high as 73.22±0.02, and the antibacterial rate of the sterile fermentation liquid of the strain at a concentration of 20% was as high as 100%, with high prevention and control efficiency, and it is green and environmentally friendly, and will not cause pollution to the environment; at the same time, the strain can also antagonize other strains, and has a wide antibacterial spectrum against tobacco brown spot pathogens, tobacco black shank pathogens, pepper phytophthora pathogens, and pepper white rot fungi, providing a theoretical basis for the subsequent research and development of antagonistic agents for other plant pathogens. Disclosed is a Bacillus amyloliquefaciens for antagonizing tobacco target spot disease pathogens, BRIEF DESCRIPTION OF THE DRAWINGS

[0013] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are only embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on the provided drawings without paying creative work.

[0014] Figure 1 The accompanying drawing is a diagram showing the results of confrontation culture between Bacillus amyloliquefaciens JQ-4 and tobacco target spot provided by the present invention;

[0015] Figure 2 The accompanying drawing is a diagram showing the antibacterial effect of Bacillus amyloliquefaciens JQ-4 provided by the present invention on the hyphae of tobacco target spot pathogen;

[0016] Figure 3 The accompanying drawing is a colony morphology diagram of Bacillus amyloliquefaciens JQ-4 provided by the present invention;

[0017] Figure 4 The accompanying drawing is a gel electrophoresis diagram of the 16S rDNA PCR product of Bacillus amyloliquefaciens JQ-4 provided by the present invention;

[0018] Figure 5 The accompanying drawing is a phylogenetic tree result diagram of Bacillus amyloliquefaciens JQ-4 provided by the present invention;

[0019] Figure 6 The accompanying drawing shows the antagonistic effect of Bacillus amyloliquefaciens JQ-4 provided by the present invention on the tested pathogenic bacteria. DETAILED DESCRIPTION

[0020] The following will be combined with the drawings in the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0021] Materials, reagents and instruments used in the examples:

[0022] Tobacco rhizosphere soil: rhizosphere soil of healthy tobacco plants collected from tobacco fields seriously infected with tobacco target spot disease in Hunan tobacco-growing areas.

[0023] Tobacco type: Yunyan 87

[0024] The test strains: tobacco target spot pathogen (Rhizoctonia solani), tobacco brown spot pathogen (Alternaria alternate), tobacco black shank pathogen (Phytophthora nicotianae), pepper phytophthora capsici Leonian and pepper white rot pathogen (Sclerotium rolfsii) were preserved in the Plant Pathology Laboratory of Hunan Agricultural University.

[0025] Culture medium and reagents

[0026] Potato glucose agar medium: Wash and peel 200g of potatoes and boil them in 1L of distilled water. After filtering through gauze, add 20g of glucose and 15-20g of agar powder. Stir with a glass rod until the medicine is dissolved, and then add distilled water to make up to 1L. Dispense 1L of medium into 500ml conical bottles, add 200ml to each bottle, sterilize it in a high-pressure steam sterilizer at 121℃ for 30min, and cool it for use.

[0027] LB liquid culture medium: weigh 10g NaCl, 10g tryptone and 5g yeast powder, add 500ml deionized water. Stir with a glass rod until all the drugs are dissolved, then add deionized water to make up to 1L, sterilize and package as above.

[0028] LB solid culture medium: Add 15-20 g / L agar powder to LB liquid culture medium and stir until the drug is dissolved. The sterilization and packaging methods are the same as above.

[0029] Reagents: glucose, agar powder, tryptone, NaCl, yeast powder, L-tryptophan (100 mg / L), indoleacetic acid (IAA) solution, purchased from Sinopharm Chemical Reagent Co., Ltd.; Salkowskis colorimetric solution, 150 mL of concentrated sulfuric acid was dissolved in 250 mL of pure water, and 7.5 mL of 0.5 mol / L ferric chloride [FeCl 3 6H 2 O].

[0030] Experimental Instruments

[0031] 2L measuring cylinder, glass rod, 2ml centrifuge tube, 15ml centrifuge tube, pipette, coating rod, disposable culture dish, high pressure steam sterilizer, clean bench, shaker, etc.

[0032] Example 1 Strain Screening

[0033] 1) Sampling

[0034] Healthy tobacco seedlings were dug up with their roots from tobacco fields seriously infected with target spot disease in Hunan tobacco growing areas. The seedlings were stored in sterilized paper bags. After being brought back to the laboratory, the soil at the roots of the tobacco was shaken off and the coarse soil was sieved to leave the fine soil.

[0035] 2) Isolation and purification of soil bacteria

[0036] Take 10g of soil sample in a conical flask containing 90mL of sterile water, shake and culture at 30℃ and 180r / min for 30min, let it stand for 5min, take the soil suspension and dilute it with sterile water to a concentration gradient of 10 -1 , 10 -2 , 10 -3 , 10 -4 , 10 -5 , 10-6 . Use a pipette to draw 0.1mL of soil dilutions of each gradient, add it to the LB solid culture medium plate for spreading and isolation, and place the coated LB culture dish in a 28℃ incubator. After 2 days, observe the growth of the colonies on the plate, and pick single colonies with different morphological characteristics for multiple purifications. Pick the hyphae of the screened fungi or make a cake at the edge of the single colony for purification. The isolated strains are numbered and stored at 4℃ for the next antagonism experiment identification. A total of 31 strains were isolated, including 22 bacterial strains, 6 fungal strains and 3 actinomycetes.

[0037] 3) Screening of biocontrol bacteria

[0038] Taking tobacco target spot pathogen (Rhizoctonia solani) as the target, the strain isolated from the rhizosphere soil of tobacco was cultured against Rhizoctonia solani. The specific steps were as follows: a 5mm punch was used to punch a bacterial cake on the edge of a PDA plate covered with tobacco target spot pathogens, and the cake was placed in the center of a fresh PDA plate as the target bacteria. The isolated bacterial colony was then inoculated about 2 cm away from the pathogen cake, and the PDA plate with only the pathogen cake was used as the control. Each was repeated 3 times, and the presence of an inhibition zone was observed after culturing at 30°C for 5 days. The mycelial diameter of the pathogen in the control group and the treatment group was measured with a vernier caliper using the cross method, and the inhibition rate was calculated. The inhibition rate of antagonistic bacteria was calculated using the following formula.

[0039] Inhibition rate (%) = (control colony diameter - treated colony diameter) / (control colony diameter - bacterial cake diameter) × 100.

[0040] Among the 22 isolated and purified bacterial strains, JQ-4, JQC-1, JQC-2, JQC-3 and JQC-4 strains had significant antagonistic effects on tobacco target spot pathogens, with inhibition rates of 73.22%, 64.55%, 62.56%, 51.91% and 52.46%, respectively (Table 1). The antagonistic effect of JQ-4 strain was significantly higher than that of other strains, with the diameter of the inhibition zone reaching 31.80 mm ( Figure 1 ).

[0041] Table 1 Antagonistic effect of antagonistic bacteria on tobacco target spot pathogen

[0042]

[0043] Note: Values ​​are mean ± standard error. Different letters in the same column indicate significant differences (P<0.05), the same below.

[0044] 4) Determination of the inhibition rate of biocontrol bacteria

[0045] The strain JQ-4 with the strongest antibacterial effect on tobacco target spot pathogen was selected for the next test. The strain JQ-4 was inoculated into 100 mL LB liquid medium and cultured at 30°C and 180 r / min for 48 h to obtain the fermentation broth (concentration 1.93×10 9 CFU / mL), centrifuged at 8000r / min for 10min, then the supernatant was aspirated and filtered with a 0.22μm bacterial filter to obtain the sterile fermentation liquid of JQ-4 strain. The sterile fermentation liquid was added to PDA cooled to about 55°C at different concentrations of 10%, 20%, and 30%, mixed evenly, and poured into a plate. The tobacco target spot pathogen cake was placed in the center of the PDA plate mixed with different concentrations of sterile fermentation liquid. Each treatment was repeated 3 times, and the control was taken without adding sterile fermentation liquid. Cultured at 30°C for 5d. The mycelial growth morphology was observed and recorded, and the inhibition rate of JQ-4 sterile fermentation liquid on tobacco target spot pathogen was calculated.

[0046] Through the inhibition test of the aseptic fermentation liquid of strain JQ-4 on tobacco target spot pathogen, it was found that different concentrations of aseptic fermentation liquid can inhibit the growth of tobacco target spot pathogen mycelium. The higher the concentration of aseptic fermentation liquid, the higher the inhibition rate ( Figure 2 , Table 2).

[0047] Table 2 Inhibition rate of JQ-4 strain sterile fermentation liquid on tobacco target spot pathogens

[0048]

[0049] 5) Identification of biocontrol bacteria strains

[0050] Morphological observation

[0051] The JQ-4 strain appears as white, nearly circular colonies on the LB plate, with wrinkles and opacity on the surface ( Figure 3 ).

[0052] Physiological and biochemical identification of biocontrol bacteria

[0053] The strains were identified physiologically and biochemically with reference to the Manual of Identification of Common Bacterial Systems, including glucose oxidation and fermentation, nitrate reduction, methyl red, VP test, starch hydrolysis reaction, gelatin liquefaction reaction determination, hydrogen sulfide production and other tests.

[0054] Physiological and biochemical characteristics showed that the starch hydrolysis, gelatin liquefaction test, and nitrate reduction test were positive, while the methyl red (MR) test, VP test, catalase, oxidase, indole reaction, and hydrogen sulfide production test were negative (Table 3).

[0055] Table 3 Physiological and biochemical characteristics of strain JQ-4

[0056]

[0057] Note: “+” in the table indicates a positive reaction, and “-” indicates a negative reaction.

[0058] Example 2 Molecular Identification of Biocontrol Bacteria

[0059] A single JQ-4 bacterial colony was picked and PCR amplified using universal primers for 16S rDNA amplification (27F: 5′-AGAGTTTGATCCTGGCTCAG-3′, as shown in SEQ ID NO.1 and 1492R: 5′-TACGGTTACCT TGTTACGAC-3′, as shown in SEQ ID NO.2). The reaction system was (50 μL): dd H 2 O (20.5 μL); 27F (1.5 μL); 1492R (1.5 μL); Mix (25 μL); DNA template (1.5 μL).

[0060] The reaction procedure was: 95℃ pre-denaturation for 5min; 94℃ denaturation for 30s; 59℃ annealing for 30s; 72℃ extension for 1.5min; 35 cycles; 72℃ extension for 10min; 4℃ storage. After the reaction was completed, the PCR product was sent to Shanghai Sangon Biotechnology (Shanghai) Co., Ltd. for sequencing. The obtained sequences were compared by BLAST nucleic acid homology on NCBI. The phylogenetic tree was established using MEGA 10.0 to determine the taxonomic status of the strain.

[0061] The 16S rDNA sequence of strain JQ-4 was amplified by PCR, and the results showed that the PCR product band size was about 1500 bp ( Figure 4 ), which is basically consistent with the expected result. The obtained sequence was compared by BLAST on NCBI, and the results showed that the sequence similarity between strain JQ-4 and Bacillus amyloliquefaciens B11 (GenBank: MF125280.1) was 99%, and the taxonomic name was Bacillus amyloliquefaciens JQ-4. JQ-4 was deposited in the China Center for Type Culture Collection on December 13, 2023, with the deposit number of CCTCCNO: M 20232552, and the deposit address is Wuhan University, Wuhan, China.

[0062] The 16S rDNA sequence of Bacillus amyloliquefaciens JQ-4 is as follows:

[0063] GGTTACCTCACCGACTTCGGGTGTTACAAACTCTCGTGGTGTGACG

[0064] GGCGGTGTGTACAAGGCCCGGGAACGTATTCACCGCGGCATGCTGATC

[0065] CGCGATTACTAGCGATTCCAGCTTCACGCAGTCGAGTTGCAGACTGCGA

[0066] TCCGAACTGAGAACAGATTTGTGGGATTGGCTTAACCTCGCGGTTTCGC

[0067] TGCCCTTTGTTCTGTCCATTGTAGCACGTGTGTAGCCCAGGTCATAAGG

[0068] GGCATGATGATTTGACGTCATCCCCACCTTCCTCCGGTTTGTCACCGGC

[0069] AGTCACCTTAGAGTGCCCAACTGAATGCTGGCAACTAAGATCAAGGGT

[0070] TGCGCTCGTTGCGGGACTTAACCCAACATCTCACGACACGAGCTGACG

[0071] ACAACCATGCACCACCTGTCACTCTGCCCCCGAAGGGGACGTCCTATCT

[0072] CTAGGATTGTCAGAGGATGTCAAGACCTGGTAAGGTTCTTCGCGTTGCT

[0073] TCGAATTAAACCACATGCTCCACCGCTTGTGCGGGCCCCCGTCAATTCC

[0074] TTTGAGTTTCAGTCTTGCGACCGTACTCCCCAGGCGGAGTGCTTAATGC

[0075] GTTAGCTGCAGCACTAAGGGGCGGAAACCCCCTAACACTTAGCACTCA

[0076] TCGTTTACGGCGTGGACTACCAGGGTATCTAATCCTGTTCGCTCCCCAC

[0077] GCTTTCGCTCCTCAGCGTCAGTTACAGACCAGAGAGTCGCCTTCGCCAC

[0078] TGGTGTTCCTCCACATCTCTACGCATTTCACCGCTACACGTGGAATTCC

[0079] ACTCTCCTCTTCTGCACTCAAGTTCCCCAGTTTCCAATGACCCTCCCCGG

[0080] TTGAGCCGGGGGCTTTCACATCAGACTTAAGAAACCGCCTGCGAGCCC

[0081] TTTACGCCCAATAATTCCGGACAACGCTTGCCACCTACGTATTACCGCG

[0082] GCTGCTGGCACGTAGTTAGCCGTGGCTTTCTGGTTAGGTACCGTCAAGG

[0083] TGCCGCCCTATTTGAACGGCACTTGTTCTTCCCTAACAACAGAGCTTTA

[0084] CGATCcGAAAACCTTCATCACTCACGCGGCGTTGCTCCGTCAGACTTTC

[0085] GTCCATTGCGGAAGATTCCCTACTGCTGCCTCCCGTAGGAGTCTGGGCC

[0086] GTGTCTCAGTCCCAGTGTGGCCGATCACCCTCTCAGGTCGGCTACGCAT

[0087] CGTCGCCTTGGTGAGCCGTTACCTCACCAACTAGCTAATGCGCCGCGGG

[0088] TCCATCTGTAAGTGGTAGCCGAAGCCACCTTTTATGTCTGAACCATGCG

[0089] GTTCAGACAACCATCCGGTATTAGCCCCGGTTTCCCGGAGTTATCCCAG

[0090] TCTTACAGGCAGGTTACCCACGTGTTACTCACCCGTCCGCCGCTAACAT

[0091] CAGGGAGCAAGCTCCCATCT, as shown in SEQ ID NO. 3;

[0092] The phylogenetic tree of strain JQ-4 was constructed with Pseudomonas cerasifera as the outgroup, and it was found that strain JQ-4 and Bacillus amyloliquefaciens were on the same branch ( Figure 5 ). Therefore, it can be preliminarily determined that strain JQ-4 is Bacillus amyloliquefaciens.

[0093] Example 3 Determination of the antibacterial spectrum of biocontrol bacteria strains

[0094] The broad-spectrum antibacterial ability of JQ-4 strain was determined by using another four pathogens preserved in the laboratory, and the mycelial growth and antibacterial effect were observed. It was found that it had a good inhibitory effect on four pathogens including tobacco brown spot pathogen and tobacco black shank pathogen, with an inhibition rate of 41.23% to 65.35% (Table 4, Figure 6 ), among which the best inhibitory effect was on tobacco target spot pathogen. It can be seen that strain JQ-4 has a broad-spectrum antibacterial activity.

[0095] Table 4 Antagonistic effect of strain JQ-4 on the tested pathogens

[0096]

[0097]

[0098] Note: Values ​​are mean ± standard error. Different letters in the same column indicate significant differences (P<0.05), the same below.

[0099] Example 4 Potted Plant Experiment

[0100] Yunyan 87 with 5 leaves and similar growth was selected for the experiment. The fermentation liquid of strain JQ-4 (1×10 9CFU / mL) as the test group, the chemical control agent was 750 times of 10% Jinggangmycin soluble powder; a 500-fold dilution made of 0.3% tetramycin aqueous solution was used as the biological control agent, and clean water was used as the blank control group. Each treatment was repeated for 3 groups with 6 tobacco plants. Each treatment was evenly sprayed on the tobacco leaves with a spray pot until the droplets just stopped dripping. The two sprayings were separated by 7 days. After the second spraying, the tobacco target spot pathogen cake (d=9mm) was inoculated on the leaf surface by needle puncture method. Each plant was inoculated with 4 leaves, and one leaf was inoculated with 4 cakes. Spray humidification was carried out once every 2 hours to ensure appropriate humidity, light from 7:00 to 19:00, dark from 19:00 to 7:00 the next day, and the temperature was 24℃. The diameter of each lesion was measured after 5 days. According to the Grading and Investigation Methods of Tobacco Pests and Diseases (GBT 23222-2008), the investigation was divided into 9 levels based on leaves. The disease index and relative control effect of each treatment were calculated: disease index = 100 × ∑ (number of diseased leaves at each level × representative value at each level) / (total number of leaves surveyed × highest representative value); relative control effect (%) = (control disease index - treatment disease index) / control disease index × 100.

[0101] The results of the greenhouse potted plant protection test are shown in Table 5. The average disease index of the treatment with JQ-4 fermentation liquid was 38.15, and the relative protection efficiency was 60.08%, which was significantly better than the clean water treatment, slightly lower than the chemical agent 10% Jinggangmycin soluble powder 750 times solution treatment, and higher than 0.3% tetramycin aqueous solution 500 times solution.

[0102] Table 5 JQ-4 control effect on tobacco target spot

[0103] deal with Disease index Effectiveness% CK 95.56 - JQ-4 fermentation broth 38.15 60.08 0.3% tetramycin 64.44 32.57 10% Jinggangmycin aqueous solution 31.85 66.67

[0104] The various embodiments in this specification are described in a progressive manner, and each embodiment focuses on the differences from other embodiments. The same or similar parts between the various embodiments can be referenced to each other.

[0105] The above description of the disclosed embodiments enables one skilled in the art to implement or use the present invention. Various modifications to these embodiments will be apparent to one skilled in the art, and the general principles defined herein may be implemented in other embodiments without departing from the spirit or scope of the present invention. Therefore, the present invention will not be limited to the embodiments shown herein, but rather to the widest scope consistent with the principles and novel features disclosed herein.

Claims

1. A Bacillus amyloliquefaciens JQ-4 antagonistic to tobacco target spot pathogen, characterized in that: The Bacillus amyloliquefaciens JQ-4 was deposited in the China Center for Type Culture Collection on December 13, 2023, with a deposit number of CCTCC NO: M 20232552, a deposit address of Wuhan University, Wuhan, China, and a taxonomic name of Bacillus amyloliquefaciens JQ-4.

2. Use of the sterile fermentation liquid of Bacillus amyloliquefaciens JQ-4 according to claim 1 in the preparation of a microbial preparation antagonistic to tobacco target spot pathogens.

3. A microbial preparation for antagonizing tobacco target spot pathogens, characterized in that: The microbial preparation comprises the sterile fermentation broth of Bacillus amyloliquefaciens JQ-4.

4. Use of the Bacillus amyloliquefaciens JQ-4 according to claim 1 in preparing a preparation antagonistic to tobacco brown spot pathogen, tobacco black shank pathogen, pepper phytophthora, and pepper white rot pathogen.

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