Aegilops sharonensis and uses thereof

Abstract

This invention discloses a type of Pantotheca acuminata and its uses, characterized by: Pantotheca acuminata (… Pantoea agglomerans After culturing FJ2 on NA medium for 24 hours, single colonies are produced. These colonies are round, pale yellow, viscous, and opaque; they are Gram-negative bacteria that move via peritrichous flagella and produce yellow pigment. They are facultative anaerobic, possessing both metabolic and fermentative chemoheterotrophic properties. The optimal temperature is 30℃. Fermentation of D-glucose and other sugars produces acid but not gas. This invention provides good control and prevention of tobacco powdery mildew and is environmentally friendly.

Description

Technical Field

[0001] This invention relates to the field of plant protection technology, specifically to a type of pantothecin FJ2, and also to the use of this pantothecin FJ2 in the control of tobacco powdery mildew. Background Technology

[0002] Tobacco white powder is a highly detrimental disease in tobacco production, caused by the fungus *[unclear - possibly a specific pathogen]. Erysiphe cichoracearum Powdery mildew (DC) is a highly infectious fungal disease with a wide host range. It occurs throughout the entire growth period of tobacco, from floating seedlings to flue-cured tobacco. In China, it is most prevalent in the tobacco-growing areas of South China, Southwest China, and Central China, with an infection rate of up to 100% and a leaf infection rate of 10% to 20%, but in severe cases, it can reach over 80%. During the vigorous growth stage of tobacco leaves, powdery mildew often causes severe damage, resulting in significant economic losses for tobacco farmers.

[0003] For a long time, chemical control agents have been the primary means of controlling tobacco powdery mildew in production. Since the 1980s, benzimidazole fungicides, represented by carbendazim, have been used in China for powdery mildew control. After more than 30 years of application, many pathogens have developed severe resistance to these agents. Subsequently, triazole fungicides, represented by triadimefon and difenoconazole, replaced carbendazim as the dominant fungicide; however, the application of triazole fungicides is limited due to concerns about crop safety. Third-generation chemical fungicides, represented by methoxyacrylates, have not been widely used for the control of tobacco powdery mildew. Because the tobacco powdery mildew pathogen has a high reproduction rate, large genetic variation, and high fitness, chemical agents are becoming increasingly difficult to control. Microbial inoculants can compete with pathogens for nutrients and space, induce plant resistance, and their secondary metabolites have inhibitory effects on pathogens and growth-promoting effects on plants. Furthermore, they are green, environmentally friendly, and safe, making them of significant application value in the control of plant diseases. Therefore, finding environmentally friendly, efficient, and safe microbial agents for the prevention and control of tobacco powdery mildew has broad development prospects and is also a necessity for the safe and sustainable development of agriculture. Summary of the Invention

[0004] The purpose of this invention is to overcome the above-mentioned problems and provide an environmentally friendly, clustered pan-mycelium FJ2 that has a good control effect on tobacco powdery mildew, a good preventive effect, and is environmentally friendly.

[0005] Another object of the present invention is to provide the use of the clump-forming pantothecin FJ2 in the prevention and control of tobacco powdery mildew.

[0006] A type of pantothecin amalgam of the present invention ( Pantoea agglomeransFJ2, morphological characteristics: After culturing on NA medium for 24 hours, it produces single colonies. The colonies are round, pale yellow, viscous, and opaque. It is a Gram-negative bacterium that moves via peritrichous flagella and produces yellow pigment. It is a facultative anaerobe and a chemoheterotrophic bacterium with metabolic and fermentative properties. The optimum temperature is 30℃. It ferments D-glucose and other sugars to produce acid but not gas. This strain was deposited on April 24, 2024, at the China Center for Type Culture Collection (address: Luojia Mountain, Wuchang, Wuhan, Hubei Province, Wuhan University), accession number: CCTCC NO: M 2024772, named *Pantotheca acuminata*. Pantoea agglomerans ) FJ2.

[0007] (a) Clumping of pan-bacteria ( Pantoea agglomerans Separation steps for FJ2:

[0008] Using the "dilution separation method," 10g of tobacco leaves were weighed into a clean beaker, and 90ml of sterile water was added. The mixture was magnetically stirred until thoroughly mixed. After standing for 20 minutes, 0.5ml of the supernatant was transferred to a 15×150mm clean test tube, and 4.5ml of sterile water was added and thoroughly mixed to obtain 10g of the supernatant. -1 The stock solution was diluted, and 0.5 ml of the supernatant was taken from the diluted test tube and 4.5 ml of sterile water was added and mixed thoroughly to obtain a 10% concentration. -2 Concentration solutions, diluted sequentially to 10 -9 Prepare soil solutions of different concentrations for later use. Take 100 µl of each of the above soil solutions of different concentrations and spread them evenly on NA plates for culturing bacteria. Incubate in the dark at 28°C. After the bacteria have grown, pick single colonies for purification.

[0009] (ii) Culture medium:

[0010] Beef extract peptone solid medium (NA): 3g beef extract, 10g peptone, 16g agar powder, 1000ml water, pH 6.8-7.2.

[0011] Beef extract peptone liquid medium (NB): 3g beef extract, 10g peptone, 1000ml water, pH 6.8-7.2.

[0012] (III) Preservation and subculturing of strains:

[0013] Use an 18×180 mm test tube, fill it with 9 ml of NA medium, make a slant, inoculate with bacteria, incubate at 28℃ for 2-3 days, store at 4℃, and subculture once every 3 months.

[0014] The purified bacteria were streaked on NA medium. After the bacteria grew, a single colony was picked and transferred to a test tube (15×150mm) containing 5 ml of NB medium with a sterile toothpick. The colony was then shaken for 24 hours. 700 µl of the bacterial culture was transferred to a 2 ml strain preservation tube. 700 µl of 30% sterile glycerol was added to the preservation tube, and the tube was stored in a freezer at -20℃ for long-term preservation.

[0015] (iv) Morphological characteristics of the strain:

[0016] The bacteria form colonies on NA plates. After 24 hours of incubation, single colonies appear. These colonies are round, pale yellow, viscous, and opaque. The bacteria are Gram-negative, motile with peritrichous flagella, and produce a yellow pigment. Based on the description in the Handbook of Bacteriological Identification, combined with Biolog identification results and 16S rDNA sequence analysis, this strain is classified as *Pantotheca cum Clumpica*. Pantoea agglomeration ) FJ2.

[0017] (v) Culture characteristics of the strain

[0018] The cultivation temperature is 20 ~ 50℃, with an optimal temperature range of 30 ~ 37℃; the initial pH of cultivation is 4.0 ~ 9.0, with an optimal pH of 6.0 ~ 7.0; the salt concentration of cultivation is 0.5 ~ 5%, with an optimal value of 0.5%; the culturable carbon sources include glucose, sucrose, fructose, galactose, lactose, maltose, cellodiol, and starch, among which glucose is the best; the culturable nitrogen sources include beef extract, peptone, KNO3, NH4NO3, (NH4)2SO4, and NaNO2, among which beef extract and peptone are the best.

[0019] Compared with existing technologies, this invention has significant beneficial effects. As shown in the above technical solutions, tobacco control experiments demonstrate that this microbial agent can significantly reduce the occurrence of tobacco powdery mildew caused by the pathogen *Tobacco Powdery Mildew*, and has a good preventative effect. This agent is prepared using wet bacteria and a bacterial culture preservation solution. The culture preservation solution consists of 0.01 mol / L phosphate buffer and 0.001 mol / L Tween 40 nonionic surfactant. This agent can be directly sprayed onto tobacco leaves. This invention is a microbial agent, environmentally friendly, and completely avoids the problems associated with the use of chemical pesticides, thus promoting pollution-free and organic production. Detailed Implementation

[0020] Example:

[0021] A type of pantothecin ( Pantoea agglomerans The separation method for FJ2 includes the following steps:

[0022] a. Isolation of the bacterial strain: Using the "dilution separation method," weigh 10g of tobacco leaves into a clean beaker, add 90ml of sterile water, stir magnetically to mix thoroughly, let stand for 20 minutes, then transfer 0.5ml of the supernatant into a 15×150 mm clean test tube, add 4.5ml of sterile water, and mix thoroughly to obtain 10... -1 The stock solution was diluted, and 0.5 ml of the supernatant was taken from the diluted test tube and 4.5 ml of sterile water was added and mixed thoroughly to obtain a 10% concentration. -2 Concentration solutions, diluted sequentially to 10 -9 Prepare soil solutions of different concentrations for later use. Take 100 µl of each of the above soil solutions of different concentrations and spread them evenly on NA plates for culturing bacteria. Incubate in the dark at 28°C. After the bacteria have grown, pick single colonies for purification.

[0023] b. Preservation and subculturing of bacterial strains: Use 18×180 mm test tubes, fill with 9 ml of NA medium, make a slant, inoculate with bacteria, incubate at 28℃ for 2-3 days, store at 4℃, and subculture once every 3 months.

[0024] The purified bacteria were streaked on NA medium. After the bacteria grew, a single colony was picked and transferred to a test tube (15×150mm) containing 5 ml of NB medium with a sterile toothpick. The colony was then shaken for 24 hours. 700 µl of bacterial culture was transferred to a 2 ml strain preservation tube. 700 µl of 30% sterile glycerol was added to the preservation tube, and the tube was stored in a freezer at -20℃ for long-term preservation.

[0025] Experimental example:

[0026] Pantothecin (clustering bacteria) Pantoea agglomerans Efficacy test of FJ2 inoculant against tobacco powdery mildew

[0027] Preparation of pathogenic fungal spore suspension: Tobacco powdery mildew specimens were collected. Conidia from individual lesions were brushed into 4℃ cold water and transferred to the upper surface of detached, disease-free tobacco leaves. The leaves were placed in a petri dish with four layers of moist filter paper and cultured at 25℃ under alternating 12h light (10000LUX) and 12h dark conditions. Subculture was performed every 9 days on the upper surface of fresh, detached tobacco leaves. To obtain a large quantity of fresh conidia for inoculation, the conidia suspension was evenly sprayed onto the upper surface of tobacco leaves using a sprayer. After 9 days of culture, dense clusters of tobacco powdery mildew conidia of similar age were produced on the upper surface of the leaves. The powdery mildew fungus was washed off the leaves with sterile water to obtain the conidia suspension. The concentration of the spore suspension was determined using a hemocytometer, and the concentration was adjusted to 10. 5 1 spore / ml, for later use.

[0028] Preparation of Pantotheca acuminata FJ2: The above-mentioned Pantotheca acuminata FJ2, used to control tobacco powdery mildew, was cultured in NB medium at 30℃ and 180 rpm with shaking for 36-48 hours. Then, it was centrifuged at 6000 rpm for 10 minutes. The wet FJ2 cells and cell preservation solution were mixed at a mass-to-volume ratio of 1:50 to prepare the inoculum. The viable cell concentration of the finished inoculum was 1×10⁻⁶. 10 -1×10 12 The bacterial culture medium was prepared using a 0.01 mol / L phosphate buffer solution containing 0.001 mol / L Tween 40 nonionic surfactant. The NB culture medium was prepared as follows: 3 g of beef extract, 10 g of peptone, and deionized water were added to 1 L. The pH was 6.8–7.2. The medium was sterilized at 120°C for 20 minutes and then dispensed for later use.

[0029] Tobacco seedlings (variety: Yunyan 87) were raised in floating seedling trays until they reached 3-4 leaves. For the biocontrol treatment group I, the above-mentioned biocontrol solution was sprayed evenly onto the surface of the tobacco leaves until it ran off; the control group was sprayed with sterile water II; for the carbendazim treatment group III, 80% carbendazim wettable powder was diluted 1000 times and sprayed until it ran off; for the difenoconazole treatment group IV, 10% difenoconazole suspension was diluted 2000 times and sprayed until it ran off. 24 hours after treatment, the above-mentioned tobacco powdery mildew spore suspension was sprayed evenly onto the leaf surface, with 12 seedlings per treatment. After inoculation, the seedlings were placed under natural light, at 15-27℃, and RH>80% for cultivation. After 24 days, the disease incidence on the leaves was observed and recorded, and the disease index was investigated to calculate the control efficacy.

[0030] Disease severity is classified according to the following disease severity grading, and the disease severity is statistically analyzed based on the affected leaf area:

[0031] Grade 0: All leaves are disease-free;

[0032] Grade 1: The area of ​​lesions accounts for less than 5% of the leaf area;

[0033] Grade 3: Lesions cover 6%–10% of the leaf area;

[0034] Level 5: Lesions cover 11%–20% of the leaf area;

[0035] Level 7: Lesions cover 21%–40% of the leaf area;

[0036] Level 9: The lesion area accounts for more than 41% of the leaf area.

[0037]

[0038]

[0039] The results of the floating seedling greenhouse experiment showed that the FJ2 treatment group of Panax notoginseng could effectively prevent the occurrence of tobacco powdery mildew, with a control effect of 82.65%.

[0040] Table 1. Efficacy of Pantotheca acuminata FJ2 in controlling powdery mildew in floating seedling tobacco seedlings 24 days after application.

[0041]

[0042] The above description is merely a preferred embodiment of the present invention and is not intended to limit the present invention in any way. Any simple modifications, equivalent changes, and alterations made to the above embodiments without departing from the technical essence of the present invention shall still fall within the scope of the present invention.

Claims

1. A type of pantothecin that clumps together ( Pantoea agglomerans FJ2, characterized in that: The preservation number of the strain is CCTCC NO: M 2024772.

2. The use of the Pantotheca floccosum FJ2 as described in claim 1 in the prevention and control of tobacco powdery mildew.

Images