CN108410778B - Method for quickly and efficiently separating bacterial ear blight bacteria of rice - Google Patents
Method for quickly and efficiently separating bacterial ear blight bacteria of rice Download PDFInfo
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Abstract
一种水稻细菌性穗枯病菌快速高效分离方法，属于微生物技术领域。其特征在于包括以下步骤：取菌悬液涂布至含氨苄青霉素的KB培养基中，25℃或40℃下培养，即可分离水稻细菌性穗枯病菌。上述的一种水稻细菌性穗枯病菌快速高效分离方法，设计合理，通过该方法能够快速、准确、有效地分离到水稻细菌性穗枯病菌，方便后续分子生物学研究。
The invention discloses a rapid and high-efficiency separation method for bacterial ear blight of rice, belonging to the technical field of microorganisms. It is characterized in that it comprises the following steps: taking the bacterial suspension and applying it to the KB medium containing ampicillin, and culturing at 25°C or 40°C, the rice bacterial ear blight can be isolated. The above-mentioned rapid and efficient separation method for bacterial ear blight of rice has a reasonable design, and the bacterial ear blight of rice can be isolated quickly, accurately and effectively by this method, which is convenient for subsequent molecular biology research.
Description
Technical Field
The invention belongs to the technical field of microorganisms, and particularly relates to rice bacterial blight bacteria (burkholderia glumae)Burkholderia glumae) A rapid and efficient separation method.
Background
Bacterial Panicle Bright of Rice (BPBR) is produced by Burkholderia glumaeBurkholderia glumaeCausing a bacterial disease. Pathogenic bacteria can produce toxoflavin and poison host plants. Mainly causes rice seedling rot, brown stain of grains and leaf sheaths (so called rice seedling rot, rice blight and glume blight), causes rice yield reduction, and the toxoflavin can also seriously affect the rice quality and harm the health of human beings and other animals.
The bacterial spike blight of rice is firstly reported in Japan, and then outbreaks of the bacterial spike blight of rice occur in various rice planting areas of Asia, America, Europe, Africa and the like, which poses serious threats to the worldwide production of rice and has attracted great attention of people. The disease has been reported in the early stage of China, and is discovered in the rice field in the central south of Taiwan province in 1984Ear blight disease; the disease is discovered in the Jiamusi area of the Heilongjiang province in 2006 by Wangchang and the like and is in the sporadic initial generation stage; rotundiflora equals 2008 that 2 rice seeds (indica rice from Hainan) in Zhejiang province are separatedB. glumaeIt is indicated that the rice species whose surface appears healthy may also carry Rhizoctonia solani. In recent years, according to the laboratory survey (unpublished data), occurrence and damage of rice panicle blight are found in rice fields from southern China, three Jiang south to southern Hainan province, and from Sichuan east to Taiwan province, in the north of China, and the occurrence area and damage of individual rice areas and years are large and serious. According to 15 villages and towns such as Lujian town and Stone town in Guangxi-Capu county and the like, which are surveyed in 10 months in 2017, most of rice panicles of the county such as Ben you 9901 ', ' Jing you Huazhan ', ' y you 1998 ', ' Lu you 1988 ', ' Lu you Huang Li ' and ' Lu you 534 ' occur to different degrees, and the occurrence area is about 500 hm2Generally, the ear disease rate is 6.5-20%, and the serious ear disease rate is 30-50% (actual survey results, which are not published yet). Therefore, it is important to establish an accurate, fast and efficient separation system for the pathogenic bacteria. Molecular biology techniques such as PCR, Real-time PCR, duplex PCR and the like are more and more widely applied in pathogen isolation and identification. However, before the identification by using the molecular biology technology, the target bacteria are not easy to separate due to the large amount of mixed bacteria.
Disclosure of Invention
Aiming at the problems in the prior art, the invention aims to design and provide a technical scheme of a method for quickly and efficiently separating rice bacterial ear blight bacteria.
The method for quickly and efficiently separating the rice bacterial ear blight bacteria is characterized by comprising the following steps of: and (3) coating the bacterial suspension into KB culture medium containing ampicillin, and culturing at 25 ℃ or 40 ℃ to separate the rice bacterial rhizoctonia solani.
The method for quickly and efficiently separating the rice bacterial ear blight bacteria is characterized in that the bacterial suspension is obtained by the following steps: taking a disease sample, disinfecting the surface of the disease sample for 15s by using 70% ethanol, washing the disease sample for 3 times by using sterile distilled water, shearing the disease sample by using a pair of sterilizing scissors, and adding 1 mL of sterile distilled water to soak for 10 min to obtain a bacterial suspension.
The method for quickly and efficiently separating the rice bacterial ear blight bacteria is characterized in that ampicillin is used as a selection antibiotic, the ampicillin can inhibit the growth of other bacteria, the ampicillin resistance of target bacteria rice bacterial ear blight pathogenic bacteria to ampicillin is high, and the ampicillin content is 50 mug/mL.
The method for quickly and efficiently separating the rice bacterial ear blight bacteria is characterized in that the culture time is 36 hours.
The method for quickly and efficiently separating the bacterial ear blight bacteria of the rice is reasonable in design, and the bacterial ear blight bacteria of the rice can be quickly, accurately and effectively separated by the method, so that the subsequent molecular biology research is facilitated.
Drawings
FIG. 1 is a graph of the growth of LMG2196 at different temperature conditions;
FIG. 2 shows LMG2196 cultured for 48 h under different temperature conditions;
FIG. 3 shows LMG2196 colonies cultured on different media at 37 ℃ for 24 h, a: KB medium; LB culture medium; c, NA culture medium; d, PDA culture medium;
FIG. 4 shows the disease standard, a-b: samples from Zhejiang Fuyang; c: guangxi Guigang samples; d, samples of Guangxi Xingsheng county;
FIG. 5 shows the bacteria separated under different temperature, culture medium and antibiotic conditions, a-d, culturing at 25 deg.C for 36 h; e-h, culturing for 36h at 40 ℃; a, e is NA (Amp-); b, f is NA (Amp +); c, g is KB (Amp-); d, h is KB (Amp +);
FIG. 6 shows Burkholderia glumae isolated from KB medium containing Amp and not containing Amp cultured at 25 ℃ for 36h, a: KB medium, Amp-; b is KB culture medium, Amp +. 1 is DL2000 Marker; 2, LMG2196 (positive control) PCR product; 3-15, separating bacteria PCR products.
Detailed Description
The present invention is further illustrated by the following examples.
Example 1: bacterial ear blight of rice (B.glumae) Separation temperatureDetermination of degree
1.1 test strains
B.glumaeThe standard strain LMG2196 was provided by professor Schwann, university of Zhejiang.
1.2. Temperature pairB.glumaeDetermination of the Effect of growth
Inoculating LMG2196 into LB liquid culture medium (yeast powder 5 g, tryptone 10 g, NaCl 10 g, distilled water to constant volume of 1L, pH 7.0), and culturing to 108 After cfu/mL of bacterial liquid, the bacterial liquid is inoculated into 3 mL of LB liquid culture medium according to the ratio of 1:1000, and the liquid is respectively placed at 16, 25, 28, 30, 35, 37, 40 and 45 ℃ for shaking culture at 200 r/min for 48 h, and 3 times of treatment at each temperature are set. The OD of each culture solution at different time periods (0, 4, 8, 12, 24, 28, 32, 36 and 48 h) was measured by a spectrophotometer (Unico 4802 UV/VIS, China)600。
1.3 analysis of results
The results of the tests are shown in figure 1,B.glumaethe growth temperature range of the bacillus subtilis is wide, the bacillus subtilis can grow at 25-40 ℃ (shown in figures 1-2), the optimal growth temperature of the bacillus subtilis is 35-40 ℃, and the bacillus subtilis is a pathogenic bacterium suitable for growing at a high temperature. The most suitable growth range of general plant pathogenic bacteria is about 28 ℃, the plant pathogenic bacteria cannot resist high temperature and are not beneficial to growth at low temperature, so that 25 ℃ or 40 ℃ can be selected as the separation temperature, the growth of bacteria and fungi which are not beneficial to growth at low temperature or cannot resist high temperature can be inhibited, and the growth of miscellaneous bacteria can be reduced, therefore, the separation method determines the 25 ℃ and the 40 ℃ as the suitable separation temperature.
Example 2: tolerance of bacterial blight of rice (b. glumae) to antibiotics
2.1 test strains
The same as in example 1.
Resistance test of glumae to different antibiotics
Ampicillin (ampicilin, Amp), Kanamycin (Kanamycin, Kan), Streptomycin (Str) and rifampin (Rifampicin, Rif) were added to the thawed and slightly cooled NA medium, respectively, so that the concentration of the antibiotics in the medium was 50 μ g/mL, and the plates were poured. And (3) respectively coating 100 mu L of LMG2196 bacterial liquid on the prepared antibiotic plates, repeating the steps for 3 times, taking the NA plate without the antibiotic as a control, simultaneously culturing the NA plate in an incubator at 37 ℃ for 48 hours, and observing the antibiotic resistance of LMG 2196.
2.3 results and analysis
By analyzing the sensitivity of LMG2196 to antibiotics, the bacteria were found to be sensitive to Kan, Str and Rif, but resistant to Amp, and able to grow normally in antibiotic culture medium containing Amp (working concentration 50 mug/mL) (Table 1), but many phytopathogens (including fungi and bacteria) were sensitive to Amp and unable to grow in Amp-containing culture medium. Therefore, certain concentration of Amp is added into the culture medium, and some mixed bacteria can be further eliminated.
TABLE 1 sensitivity of LMG2196 to antibiotics
Example 3 bacterial ear blight of rice: (B.glumae) Isolation medium selection, optimization and colony morphology culture
3.1 test strains
The same as in example 1.
3.2 protocol
Respectively preparing NA (5 g of polypeptone, 1 g of yeast powder, 3 g of beef extract, 15 g of sucrose and 17 g of agar, adding distilled water to a constant volume of 1L, and adjusting pH to 7.0), KB (20 g of peptone, K)2HPO4 1.5 g，MgSO4·7H2O1.5 g, glycerol 15 mL, distilled water to a constant volume of 1L, pH 7.2), LB (yeast powder 5 g, tryptone 10 g, NaCl 10 g, agar 16 g, distilled water to a constant volume of 1L, pH 7.0), PDA (potato 200 g, glucose 20 g, agar 15 g, distilled water to a constant volume of 1L) plates, inoculating LMG2196 with inoculating rings and streaking, culturing in a 37 ℃ incubator for 24 h, and observing the colony morphology on each plate.
3.3 test results
B.glumaeLMG2196 can grow on a variety of media, with differences in its biological properties among different media. Colonies on KB, LB, NA, PDA were all presentRound, raised, off-white, smooth edge. Better growth on KB, LB, NA plates, relatively large colonies (around 0.70 mm in diameter), with yellow media in the KB plate where colonies are dense; the LMG2196 colony aggregation area culture medium on NA is light blue, and is more obvious in light observation; the color of the medium in the colony aggregation area on LB has almost no change. LMG2196 grew slowly on PDA plates, colonies were small (0.15-0.39 mm in diameter), and the color of the medium was almost unchanged (FIG. 3, Table 2). Because LB is a general culture medium for bacteria and is more beneficial to the growth of other pathogenic bacteria, NA and KB culture media are selected as separation culture media, so that the separation accuracy can be improved.
Table 237 deg.CBurkholderia glumaeColony morphology after 24 h of culture on different media
Example 4: rapid and efficient separation of bacterial ear blight of rice
4.1 materials and methods
The test strains were as in example 1.
4.2 B.glumaeSeparation condition screening and optimization condition verification
The disease standard samples for screening and verification are from different rice regions (including 30 samples of Zhejiang Fuyang, Xiaoshan, Ningbo, Fujian Nanping, Guangdong Shaoguan, Guangxi Guigang, Nanning, Yulin, Bobai county, Xingyun county and the like) in China, as shown in figure 4, surface sterilization is carried out on the disease standard samples by 70% ethanol for 15s, sterile distilled water is washed for 3 times, the disease samples are cut into pieces by sterilizing scissors, 1 mL sterile distilled water is added for soaking for 10 min, and the inoculation loops are dipped with bacterial suspensions which are respectively dipped in NA (Amp) without Amp-) And Amp-containing NA (Amp)+) KB without Amp, KB with Amp plates were streaked, and 4 treatments were incubated at 25 ℃ and 40 ℃ for 36h, respectively, and counted using a fully automatic colony counter (Scan 1200, interperscience, france), with 3 replicates per treatment.
And identifying the separated bacteria by PCR. Conditions for the above screening after the alignment of the inoculating loopCulturing, selecting single colony of suspected Rhizoctonia solani with the same number and colony morphology, further culturing, separating, purifying, and usingB. glumaeColony PCR was performed with the specific primers glu1/glu 2. glu 1: 5-CTCTGCAACTCGAGTGCATGAGC-3; glu 2: 5-CGGTTAGAC TAGCCACTTCTGGTAAA-3, and the fragment length is about 139 bp.
4.3 analysis of results
The colony counts differed greatly on plates streaked with bacterial suspensions from the disease-sensitive samples (FIG. 4) at different temperatures, media and antibiotics (Table 3, FIG. 5). Culturing at 40 deg.C for 36h, and culturing NA (Amp)-)、NA(Amp+) 、KB(Amp-) The plate has more bacterial colonies and more mixed bacteria, some bacterial colonies are covered by fast growing mixed bacteria, and the colony of the rhizoctonia solani is less; in KB (Amp)+) The total number of colonies on the plate is small, and the number of colonies in the form of Rhizoctonia solani is also small, so that the conditions are not favorable for the growth of the bacteria. Culturing at 25 deg.C for 36h, NA (Amp)-) The total number of plate colonies and the number of mixed bacteria are more; NA (Amp)+)、KB(Amp-)、KB(Amp+) Colonies on plates were similar, but NA (Amp)+)、KB(Amp-) Relatively many colonies, only KB (Amp)+) The plates had the most colonies and the least undesired bacteria in the form of Rhizoctonia solani. Therefore, the best separation effect was obtained by underlining culture in KB medium containing Amp at 25 ℃ for 36 h.
Inoculating loop and dipping the disease-like bacteria suspension in KB (Amp)+) And KB (Amp)-) Streaking on a plate, culturing for 36h at 25 ℃, and selecting 13 smooth convex and gray single colonies for RT-PCR detection and bacterium isolation. The results show that KB media pairs without AmpB. glumaeThe separation rate was 23.1%, and the separation rate of the KB medium containing Amp was 61.5% (fig. 6), which was about 3 times that before optimization, and the separation efficiency was greatly improved. Therefore, separateB. glumaeOptionally, KB culture medium containing Amp is used, the culture is carried out for 36h at 25 ℃, and offwhite and smooth and convex single colonies are picked for PCR identification, so that the single colonies can be separated more quickly and accuratelyB. glumae。
TABLE 3 colony counts on plates under different temperature, media and antibiotic conditions
Determination and discussion of implementation rules
Taking a disease sample, disinfecting the surface of the disease sample for 15s by using 70% ethanol, washing the disease sample for 3 times by using sterile distilled water, shearing the disease sample by using a pair of sterilizing scissors, and adding 1 mL of sterile distilled water to soak for 10 min to obtain a bacterial suspension; and (3) taking the bacterial suspension, coating the bacterial suspension into a KB culture medium containing 50 mug/mL ampicillin, and culturing for 36h at 25 ℃ to separate the rice bacterial ear blight bacteria.
The study was conducted by assaying bacterial ear blight of riceB. glumaeAccording to the growth curves of the standard strain LMG2196 under different temperature conditions, the strain can grow at relatively low temperature of 25 ℃ and 40 ℃, but the temperature of 25 ℃ and 40 ℃ is not beneficial to the growth of other pathogenic bacteria and fungi, so that the interference of a plurality of mixed bacteria can be eliminated by selecting the temperature of 25 ℃ or 40 ℃ as the separation temperature of the bacterial spike blight sample of the rice. In addition, this study demonstrates for the first timeB. glumaeIs resistant to ampicillin (Amp). Bonding ofB. glumaeThe colony morphology characteristics, the sensitivity to antibiotics and the growth temperature conditions on different selective culture media establish optimized conditionsB. glumaeThe separation method comprises the following steps: KB medium containing 50 mug/mL ampicillin was cultured for 36h at 25 ℃. By applying the optimized separation method, the pathogenic bacteria of the ear blight can be accurately, quickly and efficiently successfully separated from the susceptible standard samples taken from all over the country, the ear blight is proved to be generated in China, and the prevention and control preparation of the disease is not slow.
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