Method for identifying small physiological species of tobacco angular leaf spot bacteria

Abstract

The invention discloses a method for identifying small physiological species of tobacco angular leaf spot germs, which comprises the following steps of: inoculating the angular leaf spot germs on leaves of a susceptible tobacco variety Longjiang 911, a disease-resistant tobacco variety RW and burley 21, and observing pathological reactions on the leaves of each tobacco variety; judgment is carried out according to the following physiological race judgment standards: angular spot strains which can generate brown disease spots on leaves of a susceptible tobacco variety Longjiang 911 but cannot generate disease spots on leaves of disease-resistant tobacco varieties RW and burley 21 are judged as 0 # physiological races; the angular spot strain can generate brown disease spots on leaves of an infected tobacco variety Longjiang 911, also can generate disease spots on leaves of a disease-resistant tobacco variety burley 21, but cannot generate disease spots on leaves of a disease-resistant tobacco variety RW, and the angular spot strain is judged as the No.1 physiological race. The method for identifying the physiological races of the angular leaf spot germs is simple and easy to implement, and can accurately distinguish the physiological races of the tobacco angular leaf spot germs.

Description

Technical Field

[0001] The present invention relates to the technical field of tobacco plant protection, and more specifically, to a method for identifying the physiological race category of tobacco angular leaf spot pathogen. Background Art

[0002] Tobacco angular leaf spot is one of the main bacterial diseases that harm tobacco production. Its pathogen is Pseudomonas syringae pv.augulata. Because angular leaf spot occasionally occurs in abnormal years with rainy and humid climate, it has not been taken seriously in the early stage. However, in recent years, with the increasing number of abnormal climate years and the continuous accumulation of pathogens, it has gradually become one of the main bacterial diseases in tobacco areas around the world. In China, the disease is particularly serious in the tobacco areas in Northeast China, and there are also continuous reports of diseases in tobacco areas in Yunnan and Guizhou. The angular leaf spot pathogen is generally cultured at a temperature below 28°C in the laboratory and does not proliferate when it exceeds 30°C. This also explains why it often occurs in high-latitude, high-altitude geographical conditions and rainy and rainy climate conditions. High temperature and drought are not conducive to its outbreak. In the early stage of angular leaf spot, tiny brown necrotic spots are produced on tobacco leaves, which are not easy to be observed, but the pathogen has accumulated in the tobacco cluster stage. When tobacco reaches its vigorous growth period and reaches the top, the climatic conditions for an epidemic begin to emerge, and a large-scale outbreak will occur within a few days. At this time, spraying pesticides to suppress the outbreak will have little effect, resulting in huge losses or even a total crop failure.

[0003] At present, there are few studies on tobacco angular spot disease internationally. Only a small number of studies focus on tobacco wildfire and its pathogen, Pseudomonas syringae pv.tabaci or Pseudomonasamygdali pv.tabaci, and only one physiological race has been publicly reported. At present, China does not have a unified disease resistance identification standard for distinguishing pathogen physiological races. The identification of physiological races of angular spot pathogens is a prerequisite for the next step of angular spot disease resistance breeding. There is no unified identification standard for physiological races of angular spot pathogens in China, which has caused the angular spot disease resistance breeding work to lag behind.

[0004] Therefore, it is necessary to provide a method for accurately distinguishing the physiological races of tobacco angular leaf spot pathogenic bacteria to solve the above problems. Summary of the invention

[0005] The object of the present invention is to provide a method for identifying physiological races of Pseudomonas syringae pv. tabaci, which uses known disease-resistant tobacco varieties RW, Burley 21, and the susceptible tobacco variety Longjiang 911, and cross-verifies the physiological races of Pseudomonas syringae pv. tabaci through field and greenhouse inoculation experiments. The method is simple and easy to implement, can accurately identify the physiological races of Pseudomonas syringae pv. tabaci, can be used for the discovery of new physiological races of Pseudomonas syringae pv. tabaci, and also provides an experimental basis for cultivating new tobacco varieties resistant to Pseudomonas syringae pv. tabaci.

[0006] To achieve the above object, the present invention adopts the following technical solutions:

[0007] The present invention first provides a method for identifying physiological races of Pseudomonas syringae pv. tabaci, comprising the following steps:

[0008] Inoculate Pseudomonas syringae pv. tabaci on the leaves of the susceptible tobacco variety Longjiang 911, the disease-resistant tobacco varieties RW and Burley 21, and observe the pathological reactions on the leaves of each tobacco variety;

[0009] Judge according to the following physiological race judgment criteria through the pathological reactions: A Pseudomonas syringae pv. tabaci strain that can produce brown lesions on the leaves of the susceptible tobacco variety Longjiang 911 but cannot produce lesions on the leaves of the disease-resistant tobacco varieties RW and Burley 21 is judged as physiological race 0; A Pseudomonas syringae pv. tabaci strain that can produce brown lesions on the leaves of the susceptible tobacco variety Longjiang 911, can also produce lesions on the leaves of the disease-resistant tobacco variety Burley 21, but cannot produce lesions on the leaves of the disease-resistant tobacco variety RW is judged as physiological race 1.

[0010] In a specific embodiment of the present invention, the inoculation time of Pseudomonas syringae pv. tabaci is the rosette stage or the vigorous growth stage of tobacco.

[0011] In a specific embodiment of the present invention, the observation of the pathological reactions on the leaves of each tobacco variety is carried out one week after inoculation.

[0012] In a specific embodiment of the present invention, the inoculation is spray inoculation or injection inoculation; specifically, when injection inoculation is used, an inoculation solution of Pseudomonas syringae pv. tabaci strain with a concentration of 0.001 OD is used.

[0013] In a preferred embodiment of the present invention, the injection inoculation is to puncture holes on both sides of the leaf vein, press on the position of the hole on the front side of the leaf surface, and inject the inoculation solution of Pseudomonas syringae pv. tabaci strain into the leaf from the back side of the leaf surface for injection inoculation; specifically, the injection inoculation is to use fully expanded leaves as inoculation leaves, puncture holes on both sides of the leaf vein with a syringe needle, suck the inoculation solution of Pseudomonas syringae pv. tabaci strain with a needleless syringe, press on the position of the hole on the front side of the leaf surface, and inject the inoculation solution of Pseudomonas syringae pv. tabaci strain into the leaf from the back side of the leaf surface for injection inoculation to form an injection area with a diameter greater than 1 cm.

[0014] The beneficial effects of the present invention are as follows:

[0015] Based on the inoculation pathological experiment of tobacco angular leaf spot strains, the present invention has established a method for identifying the physiological race categories of tobacco angular leaf spot pathogens. This method is simple, easy to implement, and has high accuracy. It can accurately distinguish the physiological race categories of tobacco angular leaf spot pathogens and can also discover new physiological races of angular leaf spot pathogens, providing an important basis for future tobacco angular leaf spot disease resistance breeding work. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] The following further elaborates on the specific embodiments of the present invention with reference to the accompanying drawings.

[0017] Figure 1 It is a field identification experiment for physiological races of tobacco angular leaf spot pathogens; among them, RW, B21, and LJ911 are tobacco varieties for identifying 3 physiological races of angular leaf spot pathogens, and ANG1 - ANG4 are angular leaf spot strains.

[0018] Figure 2 It is a greenhouse identification experiment for physiological races of tobacco wildfire pathogens; among them, RW, B21, and LJ911 are tobacco varieties for identifying physiological races of angular leaf spot pathogens; the inoculation positions of each strain on the leaf surface are as follows: upper left: angular leaf spot strain ANG3, middle left: angular leaf spot strain ANG1, lower left: angular leaf spot strain ANG5, upper right: angular leaf spot strain ANG2, middle right: angular leaf spot strain ANG4, lower right: angular leaf spot strain ANG6; the arrows indicate the chlorosis symptoms of angular leaf spot strains ANG3 and ANG4 (this symptom shows non - infection). SPECIFIC EMBODIMENTS

[0019] To more clearly illustrate the present invention, the following further describes the present invention with reference to preferred embodiments and the accompanying drawings. Similar components in the drawings are denoted by the same reference numerals. Those skilled in the art should understand that the content specifically described below is illustrative rather than restrictive, and should not be used to limit the protection scope of the present invention.

[0020] The disease - resistant tobacco varieties RW, Burley 21, and the disease - susceptible tobacco variety Longjiang 911 used in the following embodiments are all well - known tobacco varieties at home and abroad. Among them, the disease - resistant tobacco variety RW is recorded in Patent CN103563603B and is all preserved by the Tobacco Science Research Institute of Heilongjiang Provincial Company, China National Tobacco Corporation.

[0021] The tobacco angular leaf spot strains ANG1, ANG2, ANG3, ANG4, ANG5, and ANG6 used in the following embodiments are all obtained by the Tobacco Science Research Institute of Heilongjiang Provincial Company, China National Tobacco Corporation through bacterial isolation, purification, and identification, and are preserved by the Tobacco Science Research Institute of Heilongjiang Provincial Company, China National Tobacco Corporation.

[0022] Example 1 Field Identification Experiment for Physiological Races of Tobacco Angular Leaf Spot Pathogens

[0023] I. Cultivation of tobacco hosts for disease resistance identification

[0024] The disease-resistant tobacco variety RW can resist physiological races 0 and 1 of Pseudomonas syringae pv. tabaci; the disease-resistant tobacco variety Burley 21 (B21) only resists physiological race 0; the susceptible tobacco variety Longjiang 911 (LJ911) is used as a susceptible control.

[0025] The above three tobacco varieties are transplanted to the field after seedling raising and temporary planting. When the tobacco grows to the rosette stage or the vigorous growth stage, a Pseudomonas syringae pv. tabaci inoculation test is carried out. 48 plants of each tobacco variety are planted, among which 12 plants are inoculated with each of the 4 Pseudomonas syringae pv. tabaci strains ANG1, ANG2, ANG3, and ANG4.

[0026] II. Preparation of Pseudomonas syringae pv. tabaci strain inoculation solution

[0027] Take out the frozen bacterial solutions of the 4 Pseudomonas syringae pv. tabaci strains ANG1, ANG2, ANG3, and ANG4 from the -80°C refrigerator, and add 500 μL of each bacterial solution to a culture flask containing 500 mL of liquid LB medium. The culture flask is placed in a temperature-controlled shaker, and the bacterial solution is shaken at 28°C and 180 rpm for 16 hours to obtain the inoculation solutions of the 4 Pseudomonas syringae pv. tabaci strains ANG1, ANG2, ANG3, and ANG4 for standby.

[0028] III. Field inoculation of Pseudomonas syringae pv. tabaci strains

[0029] Each Pseudomonas syringae pv. tabaci strain is inoculated on the leaves of each tobacco variety by the spraying method. The specific method is as follows: each strain inoculation solution is diluted 5 times with distilled water and an organosilicon surfactant is added at a dilution ratio of 1:3000 to make a Pseudomonas syringae pv. tabaci strain inoculation solution. Use a sprayer to spray and inoculate the tobacco leaves in the field, with 12 replicates for each strain.

[0030] IV. Observation of pathological reactions

[0031] One week after inoculation, observe and photograph the pathological reactions of each strain on the leaves of each tobacco variety. The results are as Figure 1 shown. Brown lesions are produced on the leaves of the susceptible control variety Longjiang 911 by all 4 strains; all 4 strains cannot infect the disease-resistant tobacco variety RW; the Pseudomonas syringae pv. tabaci strains ANG1 and ANG2 can infect the disease-resistant tobacco variety B21, but ANG3 and ANG4 cannot infect the disease-resistant tobacco variety B21.

[0032] V. Result determination:

[0033] Judgment is made according to the following physiological race judgment criteria based on pathological reactions: Angular leaf spot strains that can produce brown lesions on the leaves of the susceptible tobacco variety LJ911 but cannot produce lesions on the leaves of the resistant tobacco varieties RW and B21 are judged as physiological race 0; Angular leaf spot strains that can produce brown lesions on the leaves of the susceptible tobacco variety LJ911 and can also produce lesions on the leaves of the resistant tobacco variety B21 but cannot produce lesions on the leaves of the resistant tobacco variety RW are judged as physiological race 1.

[0034] According to the above physiological race judgment criteria, the angular leaf spot strains ANG1 and ANG2 are classified as physiological race 1, and ANG3 and ANG4 are classified as physiological race 0, which is consistent with the actual situation.

[0035] Example 2 Greenhouse Identification Experiment of Physiological Races of Tobacco Wildfire Bacteria

[0036] I. Cultivation of Resistant Identification Host Tobacco

[0037] Three tobacco varieties, RW, B21, and LJ911, are transplanted into flower pots after seedling raising and temporary planting. When the tobacco grows to the rosette stage, the angular leaf spot bacteria inoculation experiment can be carried out. Four plants of each tobacco variety are planted, and four plants are inoculated with each of the six angular leaf spot strains ANG1, ANG2, ANG3, ANG4, ANG5, and ANG6.

[0038] II. Preparation of Angular Leaf Spot Strain Inoculation Solution

[0039] Take out the frozen bacterial solutions of the six angular leaf spot strains ANG1, ANG2, ANG3, ANG4, ANG5, and ANG6 from the -80°C refrigerator. Take 50 μL of each bacterial solution and add it to a culture flask containing 50 mL of liquid LB medium. The culture flask is placed in a temperature-controlled shaker, and the bacterial solution is shaken at 28°C and 180 rpm for 16 hours. Then, centrifuge at 5000 rpm for 10 minutes, pour off the supernatant, and resuspend the obtained bacterial cell precipitate with sterilized PBS solution; centrifuge again at 5000 rpm for 10 minutes, pour off the supernatant, and resuspend the obtained bacterial cell precipitate with sterilized PBS solution to prepare a bacterial solution mother liquor with a concentration of 0.1 OD. The bacterial solution mother liquor is diluted 10-fold with sterilized PBS solution to finally prepare an inoculation solution with a concentration of 0.001 OD for standby.

[0040] III. Greenhouse Inoculation of Angular Leaf Spot Strains

[0041] Inject each angular leaf spot strain onto the leaves of each tobacco variety using the injection method. The specific method is as follows: Select two fully expanded leaves at the upper leaf position of the rosette stage of each tobacco variety as the inoculated leaves. Use a 1 mL syringe needle to pierce 3 holes on both sides of the leaf veins. Use a syringe without a needle to suck up the inoculum. Press the finger on the front hole position of the leaf surface, and inject the inoculum from the back of the leaf surface into the leaf for injection inoculation to form an injection area with a diameter greater than 1 cm. Inject 6 angular leaf spot strains onto the same leaf. Inject 2 leaves for one tobacco plant, with 4 replicates for each tobacco variety, and a total of 8 replicates for each strain.

[0042] IV. Observe the pathological reaction

[0043] One week after inoculation, observe and photograph the pathological reactions of each strain on the leaves of each tobacco variety. The results are as Figure 2 shown. All 6 strains produced brown lesions on the leaves of the susceptible tobacco variety Longjiang 911; none of the 6 strains could infect the resistant tobacco variety RW; the angular leaf spot strains ANG1, ANG2, ANG5, and ANG6 could infect the resistant tobacco variety B21 (producing brown lesions), but ANG3 and ANG4 could not infect B21 (only chlorosis symptoms occurred in the inoculation area, without necrotic spots).

[0044] V. Result determination:

[0045] Make a determination according to the following physiological race determination criteria through the pathological reaction: Angular leaf spot strains that can produce brown lesions on the leaves of the susceptible tobacco variety LJ911 but cannot produce lesions on the leaves of the resistant tobacco varieties RW and B21 are determined as physiological race 0; Angular leaf spot strains that can both produce brown lesions on the leaves of the susceptible tobacco variety LJ911 and produce lesions on the leaves of the resistant tobacco variety B21 but cannot produce lesions on the leaves of the resistant tobacco variety RW are determined as physiological race 1.

[0046] According to the above physiological race determination criteria, classify the angular leaf spot strains ANG1, ANG2, ANG5, and ANG6 as physiological race 1, and classify ANG3 and ANG4 as physiological race 0. This result is consistent with the above field identification results and is in line with the actual situation.

[0047] Obviously, the above embodiments of the present invention are merely examples for clearly illustrating the present invention, rather than limiting the implementation manners of the present invention. For those of ordinary skill in the art, other different forms of changes or modifications can be made based on the above description. It is impossible to list all the implementation manners here. Any obvious changes or modifications derived from the technical solutions of the present invention still fall within the protection scope of the present invention.

Claims

1. A method for identifying the physiological race of tobacco angular leaf spot pathogen, characterized in that: The steps include: The angular leaf spot pathogen was inoculated on the leaves of the susceptible tobacco variety Longjiang 911, the resistant tobacco varieties RW and Bailie 21, and the pathological reactions on the leaves of each tobacco variety were observed. The determination is made based on pathological reactions according to the following physiological race determination standards: the angular spot strain that can produce brown spots on the leaves of the susceptible tobacco variety Longjiang 911, but cannot produce spots on the leaves of the resistant tobacco varieties RW and Bailie 21, is determined to be physiological race 0; the angular spot strain that can produce brown spots on the leaves of the susceptible tobacco variety Longjiang 911 and the leaves of the resistant tobacco variety Bailie 21, but cannot produce spots on the leaves of the resistant tobacco variety RW, is determined to be physiological race 1.

2. The method according to claim 1, characterized in that The inoculation time of the angular leaf spot pathogen is the clustering stage or the vigorous growth stage of tobacco.

3. The method according to claim 1, characterized in that The observation of pathological reaction on leaves of each tobacco variety was carried out one week after inoculation.

4. The method according to claim 1, characterized in that: The inoculation is spray inoculation or injection inoculation.

5. The method according to claim 4, characterized in that The injection inoculation is to pierce holes on both sides of the leaf veins, press the hole positions on the front side of the leaf surface, and inject the inoculation liquid of the angular spot strain into the leaf from the back side of the leaf surface for injection inoculation.

Images