CN101268744A - Method for vaccination soybean with phytophthora sojae - Google Patents
Method for vaccination soybean with phytophthora sojae Download PDFInfo
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- CN101268744A CN101268744A CNA2008101059577A CN200810105957A CN101268744A CN 101268744 A CN101268744 A CN 101268744A CN A2008101059577 A CNA2008101059577 A CN A2008101059577A CN 200810105957 A CN200810105957 A CN 200810105957A CN 101268744 A CN101268744 A CN 101268744A
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- phytophthora
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Abstract
The invention discloses a method for Phytophthora sojae to inoculate sojae. The method for Phytophthora sojae to inoculate sojae provided by the invention is a method in which the spore suspension of the Phytophthora sojae is used to inoculate a sojae seedling of which the radicel exposes out of the seed capsule. A swarm spore root inoculating method of the invention can do inoculation strictly and quantificationally, and avoids overlarge inoculum concentration to result in that the original disease resistant variety is in susceptibility, thereby ensuring to be more effective and accurate in the application of the filtration of the anti-source; at the same time, the method of the invention has the advantages of small occupied space, less labors, short experiment period and simple latter material treatment in the actual operation, therefore, the method of the invention is suitable for popularization and application.
Description
Technical field
The present invention relates to a kind of method of vaccination soybean with phytophthora sojae, particularly a kind of zoospore root inoculation method.
Background technology
Soybean phytophthora root rot (Phytophthora root rot) is by soyabean phytophthora (Phytophthorasojae Kaufmann ﹠amp; Gerdemann) infecting and cause, is to endanger one of extremely serious, destructive soybean diseases of tool, can cause the soybean total crop failure under environmental condition helps situation that disease takes place.The soybean breeding for disease resistance is the most direct, the valid approach of control soybean phytophthora root rot.Since the mid-90 in 20th century, China has carried out the beans phytophthora root rot germ plasm resource screening of the large-scale Chinese People's Anti-Japanese Military and Political College.The basic skills of resistance screening is the hypocotyl wound inoculation method that adopts Kaufmann and Gerdemann to propose.Though hypocotyl wound inoculation method is as a kind of international inoculation method comparative maturity, but its deficiency is arranged also: this method all is difficult to control to bacterium block size, bacterium amount what and cell age, the bacterium that connects than the morbidity of actual field is measured generally much bigger, such inoculum concentration pressure is excessive, general in anti-or low anti-kind may be accredited as susceptible; In addition, the disease resistance that this method is identified mainly is anti-expansion, inaccurate for anti-variety identification of invading, thereby can reduce the anti-source quantity that screens, especially the screening to horizontal disease-resistant variety is very disadvantageous, and this method causes plant death easily because of otch is excessive in operating process, can cause the mistake sieve; Because be the inoculation in seedling stage, each is handled needs 10 strains at least, 3 times are repeated is 30 strains, needs condition of pot, and it is bigger to take up room, and required time is long, and workload is bigger; Soybean phytophthora root rot is a quarantine venereal disease evil, and the experiment material amount is big, deals with time-consumingly, and risk is big.
Summary of the invention
The method that the purpose of this invention is to provide a kind of vaccination soybean with phytophthora sojae.
The method of a kind of vaccination soybean with phytophthora sojae provided by the present invention is the big bean seedlings that expose kind of skin with the zoospore suspension inoculation radicle of soyabean phytophthora.
Described method specifically can be described big bean seedlings is immersed in the described spore suspension, and then carries out common cultivation.
Wherein, for guaranteeing inoculation efficient, the radicle length of described big bean seedlings should be at least 2cm.
The radicle of described big bean seedlings immerses described spore suspension 1cm at least.
The consumption of described spore suspension can be more than or equal to 250 spore/1 big bean seedlings.
The described time of cultivating altogether can be 5-7 days.
Described temperature of cultivating altogether can be 22 ℃-24 ℃, is preferably 22 ℃.
Described light intensity of cultivating altogether can be 250-290 μ molm
-2S
-1, be preferably 270 μ molm
-2S
-1
The application of above-mentioned arbitrary method in resistance screening or pathogen virulence mensuration.
The state (the soyabean phytophthora zoospore infects soybean root under the natural conditions) that the present invention simulates field natural occurrence provides a kind of zoospore root inoculation method, this method can make hypocotyl of soybean and root all can touch zoospore, zoospore both can have been invaded from root and also can have been invaded from hypocotyl, and this is just more approaching with the mode of infection in field; Compare with hypocotyl wound inoculation method, zoospore root inoculation method can strict quantitatively inoculation, has avoided inoculum concentration excessive and cause originally that disease-resistant kind shows as susceptible proterties, make its in the application of resistance screening more effective accurately; Simultaneously the inventive method is in practical operation, has to take up room that little, the required amount of labour is little, experimental period short, the simple advantage of later experiments material processed.Of the present invention method is simple, can the short time carry out the evaluation of resistance resource in enormous quantities in the laboratory.
Embodiment
Determining of the experimental procedure of embodiment 1, zoospore root inoculation method and inoculum concentration
Present embodiment No. 1 biological strain (Xu Xiuhong of soyabean phytophthora; Lv Huiying; Qu Juanjuan; Yang Qingkai. the pathogen of soybean root rot biological strain is identified and oxicity analysis; the plant protection journal; 2003,30 (2): 125-128) be experimental strain, with farming 10 (disease-resistant varieties) and sloan (susceptible variety) the conduct experiment soybean varieties of pacifying.
Concrete operations are as follows:
(1) preparation of big bean seedlings
The sloan soya seeds is preserved moisture vernalization 2-3 days during to the long 2cm of its radicle left and right sides, select the long consistent seedling of radicle, its branch is installed in the ampoule bottle (volume 10ml) that contains equivalent sterilization running water, all be immersed in hypocotyl 1cm and root in the sterile water, cotyledon is stayed outside the bottle, and 4 strain/bottles are to guarantee in the growth enough spaces being arranged, be placed in the illumination box, at 22 ℃, 270 μ molm
-2S
-1Cultivate standby under the condition of light intensity.
The soya seeds of peaceful farming 10 is also cultivated according to the method described above, obtains the big bean seedlings of peaceful farming 10.
(2) preparation of zoospore and counting
No. 1 biological strain of soyabean phytophthora is inoculated on the carrot agar plate activation culture 3-5 days; Several piece soyabean phytophthora bacterium dish is put into aseptic sky sterilization culture dish, pour the sterilization running water into and do not have bacterium dish surface, changed the sterilization running water every 6 hours 1 time, cultivated 24-48 hour, to producing a large amount of sporangiums and discharging zoospore until just; With double gauze the soyabean phytophthora butterfly is filtered out, keep filtrate, promptly obtain the zoospore suspension of No. 1 biological strain of soyabean phytophthora.
The zoospore suspension 5 μ l that draw above-mentioned acquisition with micro sample adding appliance drag into an elongated band on clean slide, under 10 * 10 power microscope visuals field, calculate the sum of zoospore, repeated sampling 5 times, detect the concentration of zoospore in back and the diluted suspension, the concentration that makes the zoospore suspension of No. 1 biological strain of soyabean phytophthora is 1000 spores/ml suspension.
(3) zoospore is irritated the root inoculation
Draw the zoospore suspension that step (2) obtains with pipettor, be inoculated into respectively and be equipped with in the step (1) in the 10ml ampoule bottle of big bean seedlings that radicle exposes kind of skin, the big bean seedlings of 4 strains in each bottle, each soybean varieties is respectively by 10 zoospore/bottles (being that inoculum density is 2.5 spores/big bean seedlings of 1 strain), 100 zoospore/bottles (being that inoculum density is 25 spores/big bean seedlings of 1 strain), 1000 zoospore/bottles (being that inoculum density is 250 spores/big bean seedlings of 1 strain), 3000 zoospore/bottles (being that inoculum density is 750 spores/big bean seedlings of 1 strain), the inoculum density of 10000 zoospore/bottles (being that inoculum density is 2500 spores/big bean seedlings of 1 strain) is inoculated.After inoculation finishes, ampoule bottle is put to illumination box, at 22 ℃, 270 μ molm
-2S
-1Cultivated 5-7 days under the condition of light intensity.
Do not cultivate the identical time under the same conditions in contrast to inoculate peaceful agricultural 10 of any bacterium with the sloan soya seeds.
(4) identify whether inoculate soyabean phytophthora
Identify whether inoculate soyabean phytophthora by the method that resistance is identified.Inoculate back 7 days observation experiment results, comparing plant root with blank unconverted is disease-resistant for what do not connect bacterium, and plant tip of a root browning appears in plant simultaneously, fibrous root shortens and fibrous root quantity reduces, and to be what connected bacterium be susceptible.
3 repetitions are established in experiment, and the result is as shown in table 1.
The resistance performance of table 1, phytophthora zoospore variable concentrations inoculation soybean
| Zoospore quantity (individual/bottle) soybean varieties | 10 | 100 | 1000 | 3000 | 10000 |
| Peaceful farming 10 (disease-resistant varieties) | - | - | - | - | - |
| Sloan (susceptible variety) | - | + | ++ | ++ | ++ |
-asymptomatic; The slight browning of+tip of a root, fibrous root does not shorten, quantity does not reduce; ++ tip of a root browning is serious, and fibrous root shortens, quantity reduces serious
Experimental result shows, for susceptible variety, promptly can show susceptible symptom with the inoculum density inoculation of 100 zoospore/bottles, and it is the most obvious to inoculate symptom when inoculum density is 1000 spore/bottles (being 250 spores/big bean seedlings of 1 strain); For disease-resistant variety, inoculate also not reveal any symptoms with the inoculum density of 10000 zoospore/bottles.So in the practical application, can be by the concentration of 250 spores/big bean seedlings of 1 strain as standard.Adopt this inoculation method inoculation, can make all plant symptom performance unanimities in the bottle, avoided osculant problem (some plant morbidity of often occurring when inoculating with hypocotyl wound inoculation method, and other plant do not fall ill, be difficult to judge the toxicity of bacterial strain and host's disease resistance), more help postvaccinal investigation.
Embodiment 2, the application of zoospore root inoculation method in resistance screening
The used bacterial strain of present embodiment (Li Ying, Yang Mingxiu, Wen Jingzhi, the spore group of hill body virulence variation of singly moving about of 1 generation of soybean phytophthora mushroom, Chinese oil crop journal, 2007 (4): 460-465) as shown in table 2.The used soybean varieties of present embodiment is to use kind always in 9 international soyabean phytophthora differential hosts and 11 productions, and the kind catalogue sees Table 3.
Table 2, soyabean phytophthora biological strain and corresponding toxicity formula (Li Ying, 2007) thereof
| Sequence number | The toxicity formula | Biological strain | Sequence number | The toxicity formula | Biological strain |
| 1 2 3 | 1a，7 1a 3a，1b，7 | 3 | 5 6 7 | 3a，7 7 1b，7 | 15 1 2 |
| 4 | 1a，1c，7 | 4 |
Experimental technique is as follows:
One, zoospore root inoculation method
With in the table 2 except that No. 1 biological strain all the other each biological strains infect the big bean seedlings of 20 kinds shown in the table 3 respectively, wherein, the experimental technique of the big bean seedlings of each each kind of strain infection, except inoculum density was 250 spore/1 big bean seedlings, all the other methods were all with consistent described in the embodiment 1.
Cultivate the identical time under the same conditions in contrast with the big bean seedlings of the same kind of not inoculating any bacterium respectively.
With zoospore root inoculation method, carry out in the experimentation of 6 biological strains to 20 kinds, take 1m altogether at least
2The space.Prepared big bean seedlings 3-4 days, preparation zoospore suspension (comprising the bacterium cultivation) 4-7 days when carrying out spore inoculating, about 4 hours of time, is inoculated 20 soybean varieties and can be finished in 3 hours the operation of 6 biological strains.Soybean phytophthora root rot is a quarantine venereal disease evil, and the material after the test all needs sterilization treatment, and the big bean seedlings of 1680 strains are sterilized for 1 time with the 200L hot air sterilizer with 1 sterilization of autoclave of 1 18L, 18 culture dishes and 420 ampoule bottles.
Two, hypocotyl wound inoculation method
Equally, with in the table 2 except that No. 1 biological strain all the other each biological strains infect the big bean seedlings of 20 kinds shown in the table 3 respectively, be example with No. 2 big bean seedlings of biological strain inoculation sloan below, be described in detail the experimental technique of hypocotyl wound inoculation method, the method that all the other each bacterial strains are inoculated the big bean seedlings of each kind respectively is all identical therewith.
(1) seed with soybean varieties sloan places culturing pot to cultivate soil or vermiculite, and soybean plant strain launches giving birth to true leaf after about 10-12 days.
(2) No. 2 biological strains of bacterial strain are inoculated on the carrot agar plate activation culture 4-7 days;
(3) after soybean plant strain launches living true leaf, with the blade standardized wound in about 1cm place under soybean cotyledon node after the sterilization, wound depth be no more than soybean stem thick 1/3rd, get the pathogen mycelium that has medium (square of length of side 3mm) that expands after numerous and embed in the wound; After the inoculation, plastic film on the upper lid of big bean seedlings waters in film, keeps the interior relative moisture of film more than 90%, at 22 ℃, 270 μ molm
-2S
-1Cultivate under the condition of light intensity.
Cultivate the identical time under the same conditions in contrast with the sloan soya seeds of not inoculating any bacterium.
(4) identify whether inoculate soyabean phytophthora
After the inoculation, disease plant is very fast wilts, and plant fractures from inoculation position, complete stool death, and local browning only takes place in the hypocotyl wound in disease-resistant plant, the plant continued growth.Death rate 〉=70% is designated as susceptible (S), and death rate≤30% is designated as disease-resistant (R), and death rate is designated as intermediate form (I) between 30%-70%.
With hypocotyl wound inoculation method, carry out in the experimentation of 6 bacterial classifications to 20 kinds, take 10m altogether at least
2The space.Prepared plant 10-12 days, and cultivated bacterium and need 4-7 days, carry out the bacterial classification fritter when cutting apart, about 0.5 hour of time, inoculate 20 soybean varieties and can in 20 hours, finish the operation of 6 biological strains.Soybean phytophthora root rot is a quarantine venereal disease evil, and the material after the test all needs sterilization treatment, divides the autoclave of putting into 18L for 15 times to dispose at least 18 culture dishes, 140 culturing pots, the big bean seedlings of 1680 strains, soil or vermiculites etc. of this experiment.
Table 3,20 soybean varieties (being) resource are to phytophthora root rot resistance qualification result
Annotate: 1 is hypocotyl wound inoculation method; 2 is zoospore root inoculation method; S is susceptible; R is disease-resistant; I is an osculant
Three repetitions are established in experiment, and the result is as shown in table 3.The result shows, two kinds of inoculation methods resist on most variety sources/sense performance unanimity, minority is anti-/ and the sense qualification result is opposite, under the opposite situation of qualification result, all be that hypocotyl wound inoculation method is accredited as susceptible and zoospore root inoculation method is disease-resistant, compare with hypocotyl wound inoculation method, the quantity in the anti-source that zoospore root inoculation method of the present invention identifies is many nearly 20%, be that the hypocotyl wound artificial wound of making of when inoculation makes the kind that has anti-infiltration capability originally be eliminated, cause its in actual applications efficient obviously reduce.
Above result further shows, when carrying out 20 soybean varieties with zoospore root inoculation method to the resistance screenings of 6 biological strains, and 1m
2The space just can; The running water that in ampoule bottle, adds about 9ml sterilization, select the consistent seedling of growth then, 4 strain/bottles set the ampoule bottle of different disposal, these beamhouse operations can be finished in 4h, with cultured zoospore, use filtered through gauze, the concentration and the inoculum concentration of spore suspension determined in the back at microscopically, the inoculation operation can be finished in 3h, identify that to finishing general 15d just can finish from preparing seed, save time in 1 week, can more save time as many batches of inoculations than hypocotyl wound inoculation method; During the material sterilization treatment, soybean plant strain just can be handled for 1 time with the autoclave of 1 18L, and the ampoule bottle of culture dish, inoculation just can be handled for 1 time with the 200L hot air sterilizer, and can directly use next time; When carrying out 20 soybean varieties with the hypocotyl inoculation method, to use 10m at least to the resistance screenings of 6 biological strains
2The inconsistent seedling of growth need be rejected, carry out marks for treatment, carry out the hypocotyl cut wound with scalpel then, the bacterium piece is inoculated in the wound, the sterile water that dips in sterilization with absorbent cotton is clipped in the wound and preserves moisture, at last with the plant of each processing with the Polypropylence Sheet 48h that preserves moisture, these operations are finished and are wanted at least about 20h, identify that to finishing general 22d just can finish from preparing seed; During the material sterilization treatment, soil or vermiculite, seedling and culturing pot etc. that test is used will be handled tens pots at least.
Embodiment 3, screen anti-source with zoospore root inoculation method
32 soybean varieties that present embodiment is used are produce to go up kind commonly used, with in the table 2 except that No. 1 biological strain all the other each biological strains test respectively.
With in the table 2 except that No. 1 biological strain all the other each biological strains infect the big bean seedlings of 32 kinds shown in the table 4 respectively, wherein, the experimental technique of the big bean seedlings of each each kind of strain infection, except inoculum density was 250 spore/1 big bean seedlings, all the other methods were all with consistent described in the embodiment 1.
3 repetitions are established in experiment, and the result is as shown in table 4.
Table 4,32 parts of soybean varieties resource resistant to phytophthora root rot qualification results
Annotate: S is susceptible; R is disease-resistant
Experimental result shows that the resistant to phytophthora root rot resource is very abundant in the soybean varieties, all can find corresponding anti-source to different biological strains.Simultaneously show that also the different strains toxicity of phytophthora differs greatly, in 32 soybean varieties for examination, all the 6 kinds kinds that supply the prelibation sexual type all to have resistance are had 5, account for 15.6%, all susceptible has 2, accounts for 6.3%.In all 192 were done mutually, compatibility accounted for 51% mutually as 98, and non-compatibility does there are 94 mutually, accounts for 49%, illustrates that soybean resistant to phytophthora root rot resource is very abundant, has the resource of anti-a plurality of biological strains of holding concurrently.Wherein, black farming 39, close rich 51, close varietal resistances such as rich 41 and do very well, it is relatively poor to restrain agricultural 02-15 performance.Method is simple for this, can the short time carry out the evaluation of resistance resource in enormous quantities in the laboratory.
Claims (9)
1, a kind of method of vaccination soybean with phytophthora sojae is the big bean seedlings that expose kind of skin with the spore suspension inoculation radicle of soyabean phytophthora.
2, method according to claim 1 is characterized in that: described method is that described big bean seedlings are immersed in the described spore suspension, carries out common cultivation.
3, method according to claim 1 and 2 is characterized in that: the radicle length of described big bean seedlings is at least 2cm.
4, according to claim 1,2 or 3 described methods, it is characterized in that: be that radicle with described big bean seedlings immerses described spore suspension 1cm at least in the described method.
5, according to arbitrary described method among the claim 1-4, it is characterized in that: the consumption of described spore suspension is more than or equal to 250 spore/1 big bean seedlings.
6, according to arbitrary described method among the claim 2-5, it is characterized in that: the described time of cultivating altogether is 5-7 days.
7, according to arbitrary described method among the claim 2-6, it is characterized in that: described temperature of cultivating altogether is 22-24 ℃, is preferably 22 ℃.
8, according to arbitrary described method among the claim 2-7, it is characterized in that: described light intensity of cultivating altogether is 250-290 μ molm
-2S
-1, be preferably 270 μ molm
-2S
-1
9, the application of arbitrary described method in resistance screening or pathogen virulence mensuration among the claim 1-8.
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104004816A (en) * | 2014-06-10 | 2014-08-27 | 安徽省农业科学院植物保护与农产品质量安全研究所 | Experimental method for measuring indoor control effect on soybean phytophthora root rot caused by chemical agents |
| CN105165604A (en) * | 2015-10-15 | 2015-12-23 | 中国农业科学院作物科学研究所 | Method for identifying resistance of soybeans to soybean phytophthora blight and application of method in breeding |
| CN114858111A (en) * | 2022-04-18 | 2022-08-05 | 大连海宝渔业有限公司 | Method for detecting swimming distance of kelp zoospores |
-
2008
- 2008-05-06 CN CN2008101059577A patent/CN101268744B/en not_active Expired - Fee Related
Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104004816A (en) * | 2014-06-10 | 2014-08-27 | 安徽省农业科学院植物保护与农产品质量安全研究所 | Experimental method for measuring indoor control effect on soybean phytophthora root rot caused by chemical agents |
| CN105165604A (en) * | 2015-10-15 | 2015-12-23 | 中国农业科学院作物科学研究所 | Method for identifying resistance of soybeans to soybean phytophthora blight and application of method in breeding |
| CN105165604B (en) * | 2015-10-15 | 2017-08-25 | 中国农业科学院作物科学研究所 | A kind of application for identifying soybean to the method for soybean blight resistance and its in breeding |
| CN114858111A (en) * | 2022-04-18 | 2022-08-05 | 大连海宝渔业有限公司 | Method for detecting swimming distance of kelp zoospores |
| CN114858111B (en) * | 2022-04-18 | 2023-11-28 | 大连海宝渔业有限公司 | Method for detecting swimming distance of kelp spore |
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