CN101712978B - Culture medium for detecting Burkholderia glumae - Google Patents
Culture medium for detecting Burkholderia glumae Download PDFInfo
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- CN101712978B CN101712978B CN200910044034XA CN200910044034A CN101712978B CN 101712978 B CN101712978 B CN 101712978B CN 200910044034X A CN200910044034X A CN 200910044034XA CN 200910044034 A CN200910044034 A CN 200910044034A CN 101712978 B CN101712978 B CN 101712978B
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Abstract
The invention relates to measurement for biotechnique, in particular to a culture medium for detecting Burkholderia glumae. In the ulture medium for detecting the Burkholderia glumae provided by the invention, at least one of rice leaf juice and carrot juice is added to a Burkholderia glumae growing or selective separation culture medium. The culture medium for the Burkholderia glumae obviously improves the detecting rate and the detecting speed for the Burkholderia glumae and provides guarantee for resurrection culture of strains and biological safety quarantine of rice varieties.
Description
Technical field
The present invention relates to Measurement for Biotechnique, relate in particular to culture medium for detecting Burkholderia glumae.
Background technology
Paddy bacterial glume blight (Bacterial grain rot of rice) is to be caused by clever shell Burkholderia (Burkholderiaglumae); It is a kind of in recent years serious paddy rice seed-borne disease that endangers; It not only encroaches on grain; But also cause that rice seedling rots, and causes the serious underproduction and the loss of paddy rice.Seed-borne fungi is the main path of these germ long-distance communications, and the sowing disease grain that carries disease germs meets suitable onset condition, like high temperature many sunshines, and the few susceptible disease of rainfall amount.Germ can be invaded through pore and wound, and germ breeds in the plant body, in the iuntercellular diffusion, causes the parenchymatous decomposition of leaf sheath.
Later stage in the 1970's is carried out large-area industrial sprout cultivation owing to carrying out mechanize production, and the water seed and seedling rot of rice that causes B.glumae to cause is very popular, and at present should disease have become one of severe diseases of Japanese paddy rice, and has caused spread and epidemic.At present the paddy bacterial glume blight has spread to states such as country in Southeast Asia such as Indonesia, Thailand, Vietnam, Korea S, Sri Lanka, Malaysia, Philippines and South America taxi driver brother rival Asia.The Tanzania in Africa and the continent, At, Louis of the U.S. have reported that in succession this disease takes place.This disease of reports such as Shahjahan can cause 15～80% production loss in the U.S..Also there is the generation report of paddy bacterial glume blight China Taiwan nineteen eighty-three and was doing some researchs aspect cause of disease and the varietal resistance.Also once there was report China Guizhou Province that the generation of bacillary glume blight is arranged, but further confirmed.
Existing many reports that bacillary glume blights are separated and endangered of states such as Japan, Korea S, the U.S., Vietnam.Japan and Korea S have carried out more research because Burkholderia glumae is serious to the harm of paddy rice at aspects such as cause of disease and harm generations.Tushima S. in 1986; S.Wakimoto; Deng at Selective medium for detecting Pseudomonasglumae Kurita et Tabei, report in the causal bacterium of grain rot of rice one literary composition and studied the S-PG selective medium that detects Burkholderia glumae; 2000; Mitsuo K, Kiyotsugu.O etc. have reported that in New Selective Medium for Isolation of Burkholderia glumae from Rice Seeds one literary composition research is used to separate the CCNT selective medium of Burkholderia glumae; Xianglong yuan in 2004 etc. use the KBA substratum Burkholderia glumae are carried out separation and Culture in Indentification of bacterial pathogens causing panicle blightof rice in louisiana one book; And adopt Biolog microbial identification system, traditional biochemical test, pathogenic mensuration that the glume blight bacterium is identified; Luo Jinyan etc. adopt methods such as lipid acid mensuration, Biolog microbial identification system and RAPD-PCR Burkholderia glumae to be identified Liu Youxun etc. adopt the PSA substratum that phytopathogen is cultivated in paddy bacterial glume blight pathogenic bacteria isolation identification one literary composition in screening one literary composition of xanthomonas oryzae pv. oryzicola pathotype differential variety.But speed cultivation speed and do not have relevant report with the synergistic factor that increases recall rate for being used for being added into substratum.
Summary of the invention
The object of the invention is to be provided for to add in the Burkholderia glumae substratum at least a in paddy rice leaf juice (following represent with D) and the Radix Dauci Sativae juice (following represent with C), reaches that the speed of growth of Burkholderia glumae on substratum accelerated and the increase recall rate.
Culture medium for detecting Burkholderia glumae provided by the invention adds at least a in paddy rice leaf juice and the Radix Dauci Sativae juice in Burkholderia glumae growth or selective separation substratum.
The present invention finds, through adding at least a in paddy rice leaf juice and the Radix Dauci Sativae juice, can speed the speed of growth of Burkholderia glumae on substratum, the raising recall rate.
According to embodiments of the invention, when adding paddy rice leaf juice and Radix Dauci Sativae juice simultaneously, effect of enhanced sensitivity is arranged.
The paddy rice leaf juice that preferably adds according to embodiments of the invention is or/and the concentration of Radix Dauci Sativae juice is 1-5%.
The paddy rice leaf juice that adds has the effect of enhanced sensitivity equally or/and the final concentration of Radix Dauci Sativae juice greater than above-mentioned concentration, does not have disadvantageous effect, just sees from practical angle, does not need bigger concentration.Wherein, be the best with 2.5% again in the above-mentioned concentration.
The preparation method of paddy rice leaf juice and Radix Dauci Sativae juice is very simple, promptly through after smashing paddy rice leaf or Radix Dauci Sativae, through after the filtration sterilization, can obtain paddy rice leaf juice or Radix Dauci Sativae juice.It is subsequent use to be chosen in 4 ℃ of refrigerations.
Certainly, can be assembled into the substratum test kit by synergistic factor according to the present invention, use with convenient.
The present invention adopts is used for the Burkholderia glumae substratum, the recall rate that has improved Burkholderia glumae significantly with detect speed, quarantining for the Biosafety of the resurrection cultivation of bacterial classification and rice material provides assurance.
Embodiment
Following embodiment is used for further specifying of the present invention, but is not used for limiting scope of the present invention.
Embodiment 1
The extraction of synergistic factor and medium preparation.
The preparation of paddy rice leaf juice: get fresh paddy rice leaf shred the back add zero(ppm) water according to mass ratio 1: 2 after juicer squeeze the juice, 4 ℃ of refrigerations are subsequent use after the 0.45 μ m membrane filtration degerming.Hereinafter to be referred as D.
The preparation of Radix Dauci Sativae juice: after directly the fresh carrot juicer being squeezed the juice, 4 ℃ of refrigerations are subsequent use after the 0.45 μ m membrane filtration degerming.Hereinafter to be referred as C.
PS liquid culture based formulas: (growth medium a kind of)
Yeast powder 1g
Beef extract 3g
Peptone 5g
Sucrose 15g
Zero(ppm) water 1L
Regulate PH to 7.2 behind the above composition thorough mixing, every bottle of packing 50mL, subsequent use behind 115 ℃ of sterilization 15min.
Add D, C and both mixture D+C (volume ratio is 2: 1) in the PS liquid nutrient medium (growth medium) after sterilization, be prepared into liquid nutrient medium.
To sterilization back temperature is to add D, C and both mixtures (volume ratio 2: 1) of filtration sterilization in the PSA flat board about 53 ℃, pour plate, and the surface is dried subsequent use, and the PSA substratum adds the 18g bacterial agar and gets final product in the PS liquid nutrient medium.
To sterilization back temperature is D, C and both mixtures (volume ratio: 2: 1) of interpolation filtration sterilization in S-PG selectivity agar and the CCNT selectivity agar about 53 ℃, pour plate, and the surface is dried subsequent use.
The Burkholderia glumae authentication method
Adopt Biolog microbial identification system method to carry out the evaluation of Burkholderia glumae.
The employed Biolog microorganism identification of present method appearance model is BIOLOG ELX808BLG, and the microorganism identification plate is a biolog GN2 identification plate.With carrying out enrichment culture on the special-purpose BUG plate culture medium of the doubtful colony inoculation of Burkholderia glumae to Biolog identification systems; Cultivate after 24 hours for 28 ℃ ± 1 ℃; And the BUG substratum of transferring once more of the single bacterium colony on the picking BUG, its proterties is given full expression to be fit to Biolog identify.Get colony inoculation that BUG goes up second incubation to the GN/GP-IF inoculation liquid with sterilization cotton swab picking, and to regulate bacteria suspension concentration to target turbidity be 52% ± 2, and static placement 5 minutes.Inoculation regulates in inoculation liquid to the GN2 microorganism identification plate of bacteria concentration, every hole inoculation bacterium liquid 150 μ L.The microwell plate of inoculating completion is positioned over the cultivation of preserving moisture in 28 ℃ ± 1 ℃ uses Biolog mikrobe automatic identifying system to carry out identification and analysis after 24 hours.
Embodiment 2
The synergism of synergistic factor in PSA shake-flask culture base.
With the Burkholderia glumaes of 3mm transfering loop picking with 4 ℃ of 4 weeks of preservation; In the test tube that 1ml sterilization PBS is housed, smear evenly; Getting 50 μ L is seeded in the 50ml PS liquid nutrient medium that is added with different plant milk extracts in the instance 1 that final concentration is 2.5% (v/v); And the PS liquid nutrient medium that plant milk extract is not added in inoculation does positive control (CK), simultaneously with the PS liquid nutrient medium of not inoculating Burkholderia glumae as negative control, 28 ℃ ± 1 ℃ 180rpm shaking culture; Beginning to get under the aseptic condition in per 1 hour 5 PSA agar plates of bacterium liquid coating after 4 hours counts; Liquid nutrient medium continues to cultivate, and to bacterium liquid growth 24 hours, averages and log.
Get the bacterium liquid 20mL of cultivation after 12 hours to the same specification 50mL centrifuge tube of sterilizing, behind the centrifugal 20min of 10000rpm, remove supernatant, observe thalli growth situation and photograph.
Draw following test-results behind the MV (P＞0.05) of 5 parallel tests of calculating: negative control does not all grow bacterium colony; The speed of growth of having added Burkholderia glumae in the shake-flask culture base of D+C mixture and single interpolation D obviously speeds; Its colony counts also more adds to have significantly on the substratum that extracts juice before cultivating 10 hours increases, and Burkholderia glumae colony growth significance ground increases (P＜0.05) in the substratum of the more single interpolation of the substratum D of the interior D+C of interpolation of same time hybrid cytokine.(table 1)
Table 1 Burkholderia glumae is growing state in adding heterogeneity PSA liquid nutrient medium
Cultivated 12 hours, add that the centrifugal deposition that obtains of bacterium liquid in D+C mixture and the substratum that adds D is not more added and the substratum of the single C of interpolation in the deposition that obtains obvious increase is arranged.The back thalline weight of weighing is respectively: CK (1.2g); Add D (2.1g); Add D+C (2.3g); Add C (1.3g).Weighing results shows that thalli growth is more vigorous when adding D and adding in D+C to the PS liquid nutrient medium.
Embodiment 3
The synergism of synergistic factor on the PSA substratum.
4 ℃ of Burkholderia glumaes of preserving for 4 weeks are washed lawn with sterile saline, be prepared into uniform bacteria suspension, decimal dilution method is diluted to finite concentration with bacterium liquid; Get the 0.1mL coating and add the PSA flat board that concentration is D, C and the D+C of 1%, 2.5% and 5% (v/v); The agar plate that plant milk extract is not added in coating simultaneously is as positive control, simultaneously with the flat board of not inoculating Burkholderia glumae as negative control, carry out 5 parallel tests; 28 ℃ ± 1 ℃ cultivation; Observe the colony growth situation after 10 hours, per hour observe once afterwards, and record colony growth quantity.
Test-results is: negative control does not all grow bacterium colony, and under the situation of adding D and D+C hybrid cytokine, bacterium colony speeded 5 hours than the speed of growth on other two kinds of substratum on the PSA agar plate, and the interior colony growth of observing of section also has increase significantly at the same time.Be added with the bacterium colony of growing on the agar plate of D+C mixed solution to add the bacterium colony of growing on the agar plate of D many at 18 hours, colony count close (P＞0.05) during by 24 hours.In the PSA nutrient agar, add final concentration 2.5% (v/v) D+C and can increase the speed of growth of Burkholderia glumae on agar plate; And growth population has on the also more un-added substratum significance ground to increase (P＜0.05) in the section at the same time, adds 5%D (v/v) and does not have significant difference with the D+C that adds 2.5% (v/v).(table 2)
Table 2 Burkholderia glumae is growing state in adding heterogeneity PSA agar plate
Annotate: colonies typical, unit: cfu/Ml does not appear in "/" expression
Embodiment 4
The synergism of synergistic factor on S-PG and CCNT substratum.
Preserve the rice paddy seed that contains the bacillary glume blight bacterium of paddy rice 4 weeks for 4 ℃ that take by weighing 10g or 400, put into 90mL0.001% tween-phosphate buffered saline buffer (pH 7.0) low temperature (8 ℃ ± 2 ℃) and soak 4h, smash, prepare sample extracting solution with homogenizer.After sample extracting solution placed 22 ℃～25 ℃ environment 4h; Vortex mixing suspension-s 5min; Suspension-s carries out ten times of gradient dilutions; After the dilution 10 *, 100 * and 1000 * three extent of dilution be respectively put into being added with in the plate of S-PG and CCNT of synergistic factor that final concentration is 2.5% (v/v) (each plate adds 0.2mL) of having prepared, liquid is uniformly coated on the surface of agar with L-type glass stick.The agar plate that plant milk extract is not added in coating simultaneously is as positive control; And with the flat board of not inoculating Burkholderia glumae as negative control; Carry out 5 parallel tests, the colony growth situation is observed in 28 ℃ ± 1 ℃ cultivation after 10 hours; Per hour observe once afterwards, and record colony growth quantity.
Test-results: negative control does not all grow bacterium colony; The speed of growth of having added Burkholderia glumae on half selective medium of synergistic factor D+C and synergistic factor D speeded about 6 hours; Its colony counts is not added yet has significance ground to increase (P＜0.05) on the substratum of extract, it is fast and colony count is many to add on the substratum that D+C adds single D colony growth speed.(table 3)
Table 3 Burkholderia glumae different time is growing state on the selective medium that adds different components
Annotate: colonies typical, unit: cfu/mL does not appear in "/" expression
Conclusion: the present invention has obtained in two kinds of liquid medium withins plant milk extract D and the plant milk extract C that to Burkholderia glumae growth has promoter action; Through the research of adding in these two kinds of extract to agar plates is shown, when on agar plate, adding D and adding D+C growing state does not more add and the growing state of the single C of interpolation good.And growing faster with under the period; Add that to give birth to colony growth on the substratum of the more single interpolation of Burkholderia glumae growth D on the nutrient agar of D+C obvious and quantity is more; Therefore can in half selectivity nutrient agar, add plant milk extract D+C, reach the purpose of improved culture medium.The substratum of recent studies on can be used for the separation detection of paddy bacterial glume blight; Adding plant milk extract D+C final concentration is in the nutrient agar of 2.5% (v/v); Glume blight bacteria growing speed can be accelerated 5～6 hours; Can reach simultaneously the purpose of repairing impaired bacterium, improve the recall rate 30%～40% of bacillary glume blight.Add growth and selective medium that synergistic factor is applicable to Burkholderia glumae.
Claims (6)
1. substratum is used in the detection of Burkholderia glumae, it is characterized in that in Burkholderia glumae growth or selective separation substratum, adding at least a in paddy rice leaf juice and the Radix Dauci Sativae juice; Said Burkholderia glumae growth or selective separation substratum are selected from PSA substratum, S-PG substratum and CCNT substratum.
2. substratum is used in the detection of Burkholderia glumae according to claim 1, it is characterized in that the paddy rice leaf juice that adds or/and the concentration of Radix Dauci Sativae juice is 1-5%.
3. substratum is used in the detection of Burkholderia glumae according to claim 1, it is characterized in that the paddy rice leaf juice that adds or/and the concentration of Radix Dauci Sativae juice is 2.5%.
4. use substratum according to the detection of one of claim 1-3 described Burkholderia glumae, it is characterized in that described paddy rice leaf juice be meant fresh paddy rice leaf shred the back add zero(ppm) water by mass ratio 1: 2 after juicer squeeze the juice and degerming Processing of Preparation; Described Radix Dauci Sativae juice is directly the fresh carrot juicer to be squeezed the juice and degerming processing back preparation.
5. the described detection of one of claim 1-4 is with the application of substratum in detecting Burkholderia glumae.
6. test kit comprises the Burkholderia glumae growth or/and the selective separation substratum, and at least a in paddy rice leaf juice and the Radix Dauci Sativae juice; Said Burkholderia glumae growth is or/and the selective separation substratum is selected from PSA substratum, S-PG substratum and CCNT substratum.
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| 王蓟花等.《不同培养基上苜蓿假盘菌生长状况及形态学研究》.《农业科学研究》.2007,第28卷(第1期),15－17. * |
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