CN101948903B - Method for detecting whether to carry bacterial brown stripe germs in rice seed - Google Patents
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Abstract
The invention discloses a method for detecting whether to carry bacterial brown stripe germs in rice seed, comprising the following steps: a germinant rice seed is planted in sterilized pearlite; then when the rice seedling grows to 3rd-4th leaf stage, if the bacterial brown stripe symptoms happen to the rice seedling, the rice seed is judged to carry the bacterial brown stripe germs. According to the method of the invention, the rice seed is planted in pearlite and the rice seed with the bacterial brown stripe germs demonstrates corresponding symptoms after growing into the rice seedling. In addition, the detection method is simple and easy to operate.
Description
Technical field
The present invention relates to a kind of Measurement for Biotechnique, relate to a kind of rice paddy seed that detects specifically whether with the method for bacillary brown streak bacterium.
Background technology
The paddy bacterial brown streak is to bite sour bacterium oat subspecies (Acidovorax avenae subsp.avenae) by oat to cause, at first is in the news in Japan, and subsequently in the Asia, Africa, many countries in America and Europe report in succession.In China; These sick pathogenic bacterium are considered to broomcorn millet pseudomonas (Pseudomonas panici stapp) or the false unit cell (Pseudomonas syringae pv.panici) of cloves usually, but all do not see by above-mentioned two kinds of microbial paddy bacterial brown streak cause of disease probation reports.A.avenae subsp.avenae still is the pathogenic bacterium of multiple grasses such as corn, oat, barley, Chinese sorghum, sugarcane, broomcorn millet, foxtail.
The paddy bacterial brown streak is through seed-borne disease, China have only indivedual bibliographical informations A.avebae subsp.avebae cause field morbidity, so be difficult to observe the symptom in seedling stage in the field.Generally; Pathogenic bacteria can only separate on rice paddy seed and obtains; Yet other bacterium that on rice paddy seed, have many other saprophytic microorganism and false unit cells to belong to have brought very big difficulty to isolation identification, and Song W, Y etc. report that in Detection of Acidovorax avenae ssp.avenae in rice seedsusing BIO-PCR one literary composition having studied a pair of BIO-PCR primer is used to detect A.avebaesubsp.avenae; Yet; This detection technique requires the professional to accomplish, and needs valuable instrument and equipments such as PCR, and can not distinguish bacterium alive and dead bacterium.
Summary of the invention
The invention provides a kind of detection rice paddy seed simple to operation whether with the method for bacillary brown streak bacterium.
Whether a kind of rice paddy seed that detects comprises with the method for bacillary brown streak bacterium:
The rice paddy seed that germinates is cultivated in the perlite of sterilization, treat young rice seedlings growth to 3～4 leaf phases, bacillary brown streak symptom occurs, then judge the bacillary brown streak bacterium of rice paddy seed band like rice seedling.
Rice paddy seed is generally cultivated in the middle of paddy soils, and the paddy soils nutritive substance is abundant, helps microbial growth, therefore also has the mikrobe of many other kinds, has competitive inhibitory effect between the mikrobe, and paddy rice is susceptible disease not; And nutritive substance is deficient in the perlite, paddy disease-resistant property a little less than, and microbe species and comparatively small amt, paddy rice falls ill easily.
Described rice paddy seed cultivation condition is 16h on daytime, 29～31 ℃ of temperature, night 8h, 24～26 ℃ of temperature.
Described perlite size is at 2～4mm, and the humidity of cultivation is 80～90%
The present invention also provides a kind of method that detects the bacillary brown streak bacterium of rice paddy seed bacterial bearing rate, comprising:
The rice paddy seed of some germinations is cultivated in the sterilization perlite, treat young rice seedlings growth to 3～4 leaf phases,, calculate the bacterial bearing rate of the bacillary brown streak bacterium of rice paddy seed showing the rice seedling counting of bacillary brown streak symptom.This method is a rough method, but can react practical situation basically.
The inventive method through with rice paddy seed cultivation in perlite, will show corresponding symptom after growing up to seedling with the seeds of bacillary sick pathogenic bacterium, detection method is simple, and is easy to operate.
Description of drawings
Fig. 1 is the FAME lipid acid collection of illustrative plates of paddy rice brown streak bacterium typical strain R1001 of the present invention;
Fig. 2 is a paddy bacterial brown streak pathogenic bacteria 16S dendrogram of the present invention;
Fig. 3 is a paddy bacterial brown streak incidence in the different culture mediumes of the present invention.
Embodiment
The cultivation of embodiment 1 rice seedling
Rice paddy seed is soaked seed with sterile purified water, at 30 ℃ of permanent shaking tables seed soaking 24h, after be placed in uniformly and filter paper be housed (ltd provides by the fertile magnificent filter paper in Hangzhou; Diameter 9cm) in the petridish, it is moistening to add the 10ml sterile purified water, 30 ℃ of vernalization 12h; After will germinate consistent seed be seeded into respectively be equipped with perlite (in the dixie cup (diameter 7.5cm) of particle diameter 2～4mm) and paddy soils (perlite and paddy soils are all at 121 ℃ of 20min that sterilize), 3 every glass, 15 cups respectively; Be placed in the growth cabinet and cultivate, culture condition is set to: night 8h, 25 ℃; Daytime 16h; 30 ℃, treat rice seedling length to 3～4 leaf phases, find that there is the rice seedling of a large amount of similar paddy bacterial brown streak symptoms in Zhejiang in the rice varieties excellent No. 1 (agricultural company limited by shares is not forgotten in Zhejiang) in the perlite culture medium; But the paddy rice seedling in paddy soils is not found similar symptom; Other kinds [in excellent 218 (Fengle Seed Breeding Co., Ltd., Hefei), Feng You 58 (Fengle Seed Breeding Co., Ltd., Hefei), day excellent 998 (emblem, Anhui good fortune ltds of merchant farmers')] almost do not have above-mentioned symptom to take place in these two kinds of culture mediumes.Growth cabinet has passed through before cultivating paddy rice and has disinfected; Got rid of in the process that rice seedling is cultivated and received the morbific possibility of infection process; The rice seedling that a large amount of paddy bacterial brown streak symptoms is arranged in the perlite culture medium, excellent No. 1 rice paddy seed in Zhejiang has pathogenic bacteria in the explanation.
Get the sick strong junction of morbidity seedling leaves, the alcohol surface sterilization 5min with 70% washes 3 times with sterile distilled water, with sterile scissors blade is shredded and puts into the 1.5ml centrifuge tube that the 1ml sterile purified water is housed and make suspension; 180r/min shakes 3min, gets 10 μ l with pipettor then and (is the NA substratum, contains: beef extract 1g, peptone 5g in nutrient agar; Yeast extract paste 5g, sodium-chlor 5g, sucrose 10g, agar 20g; Water 1000ml) in, separates, repeat 3 times with strok method; After placing 30 ℃ of constant incubators to cultivate 48h, the picking colonies typical, after culture purified as strains tested.
The bacterial isolates that separation and purification is obtained carries out the tobacco allergometry, then with there being anaphylactoid 15 bacterial strains of typical case to be used for the pathogenic detection of paddy rice in the 24h, treats rice plant length to 3～4 leaf phases; Dip in insect needle in paddy rice stem and to get the suspension-s stab inoculation of the bacterial isolates of purifying; Entangle with plastics bag then and preserve moisture, keep relative humidity 100%, cultivate 72h at the artificial culture case; Move on to the 25 ℃ of constant temperature culture in greenhouse then; Observe disease symptom, each handles 3 plant, this experiment repetition 2 times.Observe after one week and the previous similar symptom of finding of sick seedling.
Embodiment 3 identification of strains
It is following to separate the main form of 15 strain bacterial strains and the biological property that obtain:
The thalline rod-short, bunchiness not, 1 utmost point whip of tool, size is (0.93～2.5) μ m * (0.5～0.7) μ m, and the Gram-negative reaction can be moved about, and good gas property does not produce gemma.In 28 ℃ of following growth 48h, bacterium colony is greyish white to dun, and is smoothly glossy on the NA substratum, moistening and be mucus shape, diameter 2～3mm.
The 15 strain bacterial strains that are separated to are identified through Biolog, FAME fatty acid analysis (as shown in Figure 1), 16S rDNA sequence alignment and Aaaf5 or Aaaf3/Aaar2PCR specificity.
The Biolog qualification result shows that the bacterial strain that is separated to all is accredited as A.avenae subsp.avenae, and similarity is between 0.65～0.83; The lipid acid qualification result is consistent with Biolog, and similarity is between 0.58～0.71; Typical 4 bacterial strains are carried out 16S order-checking, and sequence is submitted to the GenBank DB, corresponding sequence number is respectively HM240856～HM240859, shows 4 typical strains and the ATCC19882 of mensuration through the sequence alignment result
T(sequence number is DQ360414) similarity is 99%; Quote the amplification of Aaaf5 or Aaaf3/Aaar2 primer PCR, can produce the specific band of A.avenaesubsp.avenae.
Recently; Schaad etc. are at Reclassification of subspecies ofAcidovorax avenae asA.Avenae (Manns, 1905) emend., A.cattleyae (Pavarino; 1911) comb.nov.; A.citrulli (Schaad et al., 1978) comb.nov. proposes a novel species in and proposal ofA.oryzae sp.nov. one literary composition; The A.avenae subsp.avenae that separates on paddy rice is renamed the oryzae into Acidovorax, but this categorizing system does not also obtain final affirmation.We separate the pathogenic bacteria obtain from paddy rice and separate the A.avenae subsp.avenae similarity that obtains from seeding corn and other crops very high; So our the most above-mentioned strain separated is accredited as oat and bites sour bacterium oat subspecies (A.avenae subsp.avenae), i.e. paddy bacterial brown streak bacterium.
The detection of embodiment 4 seed-borne fungi rates
1) separation of bacterium
During 100 of picked at random are above-mentioned Zhejiang excellent No. 1 (agricultural company limited by shares is not forgotten in Zhejiang) with in the rice paddy seed of excellent 218 (Fengle Seed Breeding Co., Ltd., Hefei) kind, with mercuric chloride surface sterilization 8min, wash 2 times with sterile purified water then; Then every seed is ground, put into respectively and be numbered 1～100 the 1.5ml centrifuge tube that is added with the 1ml sterile purified water, 180r/min shakes 3min; Getting 200 μ l with pipettor then (is the NA substratum, contains: beef extract 1g, peptone 5g in nutrient agar; Yeast extract paste 2g, sodium-chlor 5g, sucrose 10g; Agar 20g, water 1000ml) in, evenly be coated with spreader; After placing 30 ℃ of constant incubators to cultivate 48h, the former mushroom of picking and brown streak is bacterium colony seemingly, after culture purified as strains tested.
2) extraction of DNA
With above-mentioned strains tested 1～2 ring of transfering loop difference picking, place the 1.5ml centrifuge tube, add the NaOH 1 μ l of 1mol/L then respectively, 2% SDS (sodium cetanesulfonate), 2.5 μ l, ddH
2O16.5 μ l, 95 water-bath 15min add 180 μ l ddH then
2O, be saved in-20 ℃ for use.
3PCR detects
The result shows: in excellent No. 1 100 seeds in middle Zhejiang, have on 31 seeds to be separated to bacterium through pcr amplification, can produce specific band, promptly have 31% seed to have the brown streak pathogenic bacteria.Yet in do not detect the brown streak pathogenic bacteria in 100 seeds of excellent 218.
In above-mentioned Zhejiang excellent No. 1 with in excellent 218 rice paddy seeds soak seed with sterile purified water, 30 ℃, the 160r/min 24h that soak seed is placed in uniformly then and filter paper is housed (irrigating magnificent filter paper ltd by Hangzhou provides; Diameter 9.0cm) in the petridish, it is moistening to add the 10ml sterile purified water, 30 ℃ of vernalization 12h, and the consistent rice paddy seed of selecting to sprout evenly is seeded into seedling dish (long 45.1cm; Wide 30.3cm, high 5.4cm) in, culture medium is used perlite and paddy soils respectively, and each sows 200; The seedling dish is placed on growth cabinet, night 8h, 25 ℃, daytime 16h; 30 ℃ of cultivations, five days " Invest, Then Investigate "s also write down incidence, this experiment repetition 2 times.
The result shows: the excellent No. 1 rice pathogenesis rate in Zhejiang is 25% in the pearlite interstitial substance, in the sickness rate of excellent 218 paddy rice be 0, two rice varieties are not all observed obvious disease symptom in paddy soils matrix.Meanwhile we have carried out separation and the evaluation of pathogenic bacteria to the excellent No. 1 morbidity rice seedling in middle Zhejiang in the pearlite interstitial substance, confirm that these morbidity rice seedlings are microbial by the paddy bacterial brown streak.
According to embodiment 4, in do not detect the existence of brown streak bacterium in excellent 218 rice paddy seeds, excellent 218 rice paddy seeds are not with the brown streak bacterium in the explanation.In excellent 218 in perlite and two kinds of matrix of paddy soils, all do not observe tangible disease symptom, prove further that the paddy bacterial brown streak is to have the brown streak bacterium because of seed in a large number in perlite, rather than extraneous pathogen infection causes.
According to embodiment 4, middle Zhejiang is excellent to have for No. 1 31% seed have the brown streak pathogenic bacteria, yet the middle Zhejiang sickness rate of excellent No. 1 paddy rice in pearlite interstitial substance is 25%, and promptly our pearlite interstitial substance is 80.6% to the recall rate of rice paddy seed band brown streak bacterium.
Claims (4)
1. one kind is detected rice paddy seed whether with the method for bacillary brown streak bacterium, comprising:
The rice paddy seed that germinates is cultivated in the perlite of sterilization, treat young rice seedlings growth to 3～4 leaf phases, bacillary brown streak symptom occurs, then judge the bacillary brown streak bacterium of rice paddy seed band like rice seedling;
Wherein, described cultivation humidity is 80～90%.
2. method according to claim 1 is characterized in that: described rice paddy seed cultivation condition is: daytime 16h, 29～31 ℃ of temperature, night 8h, 24～26 ℃ of temperature.
3. method according to claim 1 is characterized in that: described perlite size is 2～4mm.
4. method that detects the bacillary brown streak bacterium of rice paddy seed bacterial bearing rate comprises:
The rice paddy seed of some germinations is cultivated in the sterilization perlite, treat young rice seedlings growth to 3～4 leaf phases,, calculate the bacterial bearing rate of the bacillary brown streak bacterium of rice paddy seed showing the rice seedling counting of bacillary brown streak symptom.
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Non-Patent Citations (4)
| Title |
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| 刘华招.水稻机插中苗育秧基质的研究.《现代化农业》.2009,(第356期),1-3. * |
| 徐丽慧.水稻细菌性谷枯病菌的分子检测及细菌性褐条病病原鉴定研究.《中国优秀硕士学位论文全文数据库农业科技辑》.2008,(第9期),D046-29. * |
| 徐丽慧等.水稻细菌性褐条病病原的鉴定.《中国水稻科学》.2008,第22卷(第3期),302-306. * |
| 李俊平.水稻细菌性褐条病的发生及防治.《贵州农业科学》.2003,第31卷(第2期),34-36. * |
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