CN86105904A - Rhizobium inoculants - Google Patents
Rhizobium inoculants Download PDFInfo
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- CN86105904A CN86105904A CN198686105904A CN86105904A CN86105904A CN 86105904 A CN86105904 A CN 86105904A CN 198686105904 A CN198686105904 A CN 198686105904A CN 86105904 A CN86105904 A CN 86105904A CN 86105904 A CN86105904 A CN 86105904A
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Abstract
What disclosed is a kind of method that produces the new bacterial strain of the root nodule bacteria that leguminous plants uses, and this bacterial strain was not only competitive but also high fixed nitrogen feature arranged.Present method comprises at first separating and differentiating natural dominant bacterial strain at the relevant root nodule bacterium bacterial classification in specific place makes this bacterial strain mutagenesis to produce the mutant strain that can keep competitiveness and have the increase nitrogen fixing capacity then.
Description
The present invention relates generally to be used for the inoculum of leguminous crop, and relate to particularly generation can biologically competitive and have high nitrogen fixing capacity new rhizobium strains method and produce the method for these bacterial strains.
Leguminous plants and to make the phenomenon of the symbiotic relationship between the root nodule bacterium of its root dross be well-known.The bacterium of existence in the root nodule of leguminous plants root be fixed nitrogen from airborne nitrogen directly, thereby nitrogen is converted to nitrogenous compound useful on biology, this process is not only proteinic synthetic but also make and grew fabaceous soil fertile soil to carry out for nitrogen to plant, and this is used by the crop that nitrogenous nutrition is stayed after being provided with in the soil.It is pea, broad bean, alfalfa, red clover, butch clover that energy and root nodule bacterium have the fabaceous example of symbiotic relationship, common vetch and plumage French beans.
Because many plants can not be directly or direct fixed nitrogen from air indirectly on agricultural for other, thereby generally the nitrogenous fertilizer of its dependence is introduced enough nitrogen contents so that the output that obtains quite generally makes leguminous plants and non-leguminous plant crop rotation so that the nitrogen content in the soil is promoted on very economical ground in each planting time in important farming region in soil.This wide-spread with the celebrated practice of shift of crops.Moreover some leguminous plantss, particularly soybean, pea, broad bean and alfalfa itself be to can be used as important cash crop, and the growth of these crops can easily provide abundant combined nitrogen or fixed nitrogen.
Exist the bacterial classification of a large amount of root nodule bacterium (Rhizobium), and different root nodule bacterium bacterial classifications can only form symbiotic relationship and generate root nodule at the root of specific leguminous crop.For example, soybean nodulation bacterium (R.japonicum) makes the root dross of soybean, trifolium root nodule bacteria (R.trifolii) makes trefoil root dross, alfalfa Phylloxera bacterium (R.meliloti) and alfalfa and the growth of sweet clover symbiosis ground, pea root nodule bacterium (R.leguminosarum) is used for pea and common vetch, and plumage French beans root nodule bacteria (R.lupini) is used for the root dross that plumage French beans and Kidney bean root nodule bacteria (R.phaseoli) make common Kidney bean.Thereby very common way is to inoculate on soil or fabaceous seed with corresponding root nodule bacterium microbial strain culture in the agricultural practice of commodity.Usually by being coated in before planting on the seed, bacterium being sprinkling upon on the seed of plantation or spreading inoculum by in the ditch of plantation leguminous seeds, loosing and inoculate.
When producing and prepare rhizobium inoculants, preferably the contained rhizobium strains of inoculum has stronger nitrogen fixing capacity so that help the growth of plant and regulate soil.Thereby the method for the nitrogen fixing capacity of relevant improvement rhizobium strains has been carried out a large amount of research logically.The rhizobium inoculants that great majority can have been bought now comprises the selected bacterial strain with high nitrogen fixing capacity from natural group.
Yet nitrogen fixing capacity can not guarantee the survival rate of bacterium in the field.Because leguminous plants has been planted many generations in most of in the world important farming regions, many inherent rhizobium strainss are freely being survived in important farming region now.And this bacterium may not be all to be inherent natively to all these zones, but the bacterial strain of introducing breeding in many these zones, and is freely surviving because of them, and they can be considered to is inherent actually.
In soil or in the elsewhere, all bacteriums that freely surviving all are subjected to the pressure of environment continuously, can natural birth change differently, select to make it further this environment to be had adaptability and competitiveness by the pressure of the environment of their occupied ecology.Certainly, the inherent rhizobium strains just lives through these pressure.Thereby the many soil in the farming region contain now be evolved into the inherent rhizobium strains that is suitable for survival in competition existence in this specific edatopes.Thereby, even the farmer introduces the rhizobium strains with high nitrogen fixing capacity in the field, but can not guarantee that the rhizobium strains of being introduced can prevail on fabaceous.The inoculum bacterial classification of introducing promptly with in soil inherent make the bacterial classification of plant dross enter the ecologic competition state, and often all be that the inherent flora is preponderated because they are selected by the ecological pressure of competition in the environment of this field or climatic province.Thereby, when not competing, can make effectively the leguminous plants dross and effectively fixed nitrogen high nitrogen-fixing rhizobia bacterial classification in addition may be invalid inoculum in the field, this is that the local intrinsic bacterial classification that it may not had best nitrogen fixing capacity overwhelms because in fact the bacterial strain of introducing may can not make fabaceous root propagation.Relevant being created in the field is competitively to have the desirability or the method for the new rhizobium strains of high nitrogen fixing capacity not to mention in known existing document again.
Known in the past that leguminous plants root transudate can be used for making the root nodule bacterium cell development more can be in conjunction with the position of external source coagulin.People know that also this class transudate can be used as pre-treatment, can promote and strengthen the dross ability of root nodule bacterium when being inoculated on the leguminous plants with box lunch.
The present invention can be summarized as follows: the method that produces the new bacterial strain of the root nodule bacteria that can be used as the legume inoculation agent in the geographic area of selecting is to isolate many root nodule bacterium cultures from this geographic area; Identify dominant natively root nodule bacterium bacterial classification in this geographic area by culture being compared analyze; Make the culture of natural dominant bacterial strain be subjected to the processing of mutagenic compound; With in the bacterium of mutagenesis, select the mutant strain that can have high nitrogen fixing capacity when combining with leguminous plants.
The present invention is also by using this method to reach the target that produces novel bacterial.
The purpose of this invention is to provide a kind of method that produces new rhizobium strains, this bacterial strain can have highly natural competitiveness and have the enhanced nitrogen fixing capacity in the geographic area of selecting.
Another object of the present invention provides with the selected bacterial strain of this method, and this bacterial strain is more predictably promoted the growth of leguminous crop than present getable rhizobium inoculants bacterial strain.
The invention is characterized in the race condition that can in general specific agricultural environment, select the competitiveness of bacterial strain and need in the manual control environment, not simulate relevant bacterial isolates, and this simulation to be proved to be extremely difficult perfect in practice.
Other purposes of the present invention, advantage and feature can obviously be found out from following detailed description.
Fig. 1 is in order to producing according to program of the present invention, separate and the competitive rhizobium strains that identifies in the methodological schema of high azotobacter strain;
Fig. 2 for diagram from one by the graphic representation of the enhancing degree of the nitrogen fixation activity that dominant bacterial strain obtained natively of mutagenesis;
Fig. 3 represents the graphic representation of the enhanced nitrogen fixation activity that obtained from second bacterial strain;
Fig. 4 is increased the graphic representation of situation by the resulting dross of inoculum pre-treatment for expression;
The practice system that produces the method for new rhizobium bacterial classification according to the present invention carries out through series of steps. At first, need to from selected geographic area, isolate many rhizobium cultures, then it be analyzed to identify the natively dominant bacterial strain of rhizobium. The natural dominant bacterial strain that then this is identified stands the bacterium colony that the mutagenesis of the high fixed nitrogen of energy is selected in mutagenesis. After finding high fixed nitrogen mutant strain, mutant strain is turned back to the field test to determine that they can keep competitiveness can keep again sufficiently high nitrogen fixing capacity to increase output in the field. In order to fully understand the details of each program, each is discussed to it in order.
Separate the method system of intrinsic rhizobium culture from differentiating competitive bacterial strain. Opposite with the previous strategy that produces rhizobium inoculants, needs are not made an effort and are attempted to choose seeds or manage for the competitiveness in the condition of actual field according to the present invention. But the first step is differentiated those intrinsic natively dominant bacterial strains that have been present in the legume of selecting in selected geographic planting zone territory. For example select soybean as a kind of legume, people should select wherein to grow the place that soybean is arranged and differentiate in this place natural dominant intrinsic rihizobium japonicum. As used herein such, term " intrinsic " did not also mean that before the mankind occur just spontaneously, because for example rihizobium japonicum is the bacterial classification of introducing on the Western Hemisphere, but was the bacterial strain of growing up strong and sturdy natively in the soil of farming region now. The intrinsic local bacterial strain that the natural rihizobium japonicum that exists is for example now arranged in the most of soils in the Soybean production district of the U.S., and this bacterial strain has developed in the various soil and each climatic province that existence is this country.
To competitive bacterial strain determine and differentiate be at first from the legume cultivation area that has been selected the large scale collection root nodule set about. Do not determine and take over the legume field of planting with any rhizobium inoculants. Pull out crop and the root of the crop from be grown in these fields is gathered in the crops root nodule from Tanaka. Root nodule is carried out surface sterilizing, smash then in order to can obtain wherein rhizobium bacterium colony. The cell that will obtain from the root nodule of smashing is coated on the gelose medium flat board that suitable growth medium is nutrition, and cultivates in the laboratory. Then these bacterial strain bacterial strains are differentiated Continue after the process to go on.
The discriminating of bacterial isolates is a kind of just in developing technology now. Now most of bacterial isolateses are differentiated and classified and be based on the result of series of experiments. This test comprises observes morphology, bacterium serology, the quantitative susceptibility of different strains, the phage-sensitive degree of bacterial strain and the protein profiling that is produced by bacterial strain of measuring with unidirectional (one dimension) or two-way (two dimension) polyacrylamide gel electrophoresis. Any or these all technology can should be used for strain identification together. The applicant used herein and be considered to simple and easy and effective, best of breed, the most useful technology is unidirectional gel electrophoresis.
Best, from the locality of relevant crop, collect a large amount of root nodules and obtain a large amount of rhizobium cultures, for example, the culture that obtains the rhizobium strains of hundreds of (if not in thousand words) from specific geographic area and specific crop such as soybean is suitable. Then these thousands of bacterial strains are analyzed with gel electrophoresis. Analyze then the collection of illustrative plates of gel electrophoresis gained. The purpose of this operation is to determine at regnant those collection of illustrative plates of this growth district to characterize naturally dominant bacterial strain. Easy speech, a large amount of gel flat boards is analyzed and is enrolled in each group of reflection gel pattern marshalling. Relatively divide then the number of the culture in each marshalling to determine that group or that bacterial strain are most representative, can consider like this in the specific region, to select reigning bacterium. Be defined as identical bacterial strain with being found to be those separators with same gel pattern. As if can find frequent isolated different strains they have different competitiveness in different growth districts, and some bacterial strain occupies ascendancy in the growth territory of a part.
In case should be with this bacterial strain as naturally playing dominating role and competitive bacterial strain concerning this program when determining in specific growth district the most extensive isolated bacterial strain. In order fully to confirm its competitiveness, and guarantee when cultivating this bacterial strain in the past not through variation, then be suitable for the field of carrying out bacterial strain test to guarantee its suitably dross and in the environment of actual field its competitiveness can recur. The test of making nitrogen fixation activity also is suitable. This step can be carried out by the young plant in pot or basin in greenhouse or seedling rearing room. With being considered to the most competitive bacterial strain pair This plant inoculates, and makes then the mensuration of second piece reduction. This not only confirms the nitrogen fixation activity with the associated bacterial strain of crop of selecting, and as the used baseline of effort of selecting to have increased later on nitrogen fixation activity.
To emulative validation test is to test about the field of dross vigor.This has by plantation and is considered to natively the soil of dominant competitive bacterial strain and realizes.Contrast should be to comprise the seed that does not connect bacterial classification and comprise plant with the inoculation of the commercial but competitive difference of supposition.For the dominant natively competitive bacterial strain that makes success is recognized, selected bacterial classification should be able to reclaim from the root nodule the vaccinated plant, its rate of recovery should be significantly higher than never inoculation or with the rate of recovery in the plant of the inoculation of uncontested property, and this rate of recovery also should be significantly higher than the rate of recovery of the commercial bacterial strain that can reclaim from root nodule.
In order to test, can be determined to be in dominant natively and competitive bacterial strain under the condition of field with high frequency isolated again bacterial classification from fabaceous root nodule with relevant bacterial classification inoculation.Thereby to have separated with the bacterial strain that identifies in this grade program be inherent but stand to introduce once again after separate in the laboratory and can also keep its competitiveness.In addition because we have confirmed to make after this inoculation the dross of plant to increase with this step, even show that also not strengthening nitrogen fixation activity also can obtain more root nodule by using this bacterial strain.
Next step is to set about making reigning natively bacterial classification to carry out mutagenesis.Make the culture of bacterial strain be subjected to mutagenic compound such as the width of cloth is penetrated or the processing of chemical reagent.The mutagenic compound that preferentially use in this test are the chemical mutagen nitrosoguanidine.Bacterium after will handling with mutagenic compound shifts and allows and grows up in the liquid medium within and shift several times.The mutagenic strain nutrient solution is layered on the agar plate so that obtain one bacterium colony then.In case establish one bacterium colony then again with this colony lift in liquid nutrient medium and make its growth.The sample that each can be grown bacterium colony then is inoculated in the serum bottle that the leguminous plants seedling is housed.Make that seedling and root nodule bacterium are common to grow up and this seedling and environment are isolated, to detest the transparent vessel of bacterium, grow under the condition that leguminous plants grows promoting then as lid.After after a while, to carry out nitrogen fixation activity and measure, this mensurations is followed once more with vapor-phase chromatography or other similar approach and is reduced vigor as the acetylene of test seedling.Compare the separable offspring who goes out to have the mutagenesis of stronger nitrogen fixation activity by the baseline that the nitrogen fixation activity of mutant bacterial strain and dominant bacterial strain are natively used.The field test that can make bacterial strain turn back in the culture then and grow out and add to provide enough quantity to do with most promising nitrogen fixation activity.
In case identified high fixed nitrogen candidate's mutant strain, must analyze these bacterial strains then once more to guarantee can not lose natural competitiveness through mutation process.Thereby all candidate strain must regrow out and must do single competitive test in the test of actual field.Simultaneously, in greenhouse or seedling rearing room, can recur the fixed nitrogen test-results, so that guarantee can be repeatedly and strengthen nitrogen fixing capacity reliably.Owing to recur the relation of skill state of the technology of competition and field condition in greenhouse or seedling rearing room condition, competitiveness can only could be real definite in the test of field.Make bacterial strain grow and be inoculated in the leguminous plants that is fit in the plant growth zone like this.Can be at those root nodules of breeding with definite those bacterial isolateses at the number of the root nodule of quarterly inspection in plant then.For keeping competitive and realize those bacterial strains of enhanced nitrogen fixation activity, those bacterial strains should be present in that the bacterial classification of the comparable uncontested property of its quantity increases among the root nodule.In addition, comprising the field of the mutant strain with high nitrogen fixing capacity and emulative advantage should be than not having inoculum maybe can obtain other the bacterial strain of commercial uncontested property and those fields of inoculum can provide higher output.
It below is detailed description to embodiment.
Determined to produce the novel bacterial that is particularly useful as the soybean nodulation bacterium that the soybean inoculation thing in two main soybean production areas of the U.S. uses.As the first step of this method, set about making great efforts to differentiate at selected Iowa and the natural dominant bacterial classification of the soybean nodulation bacterium among the Na Zhou of Xi'an, Louis.Field team inspects in each state, does not wherein use the soybean field of commercialization inoculum between planting season.Soybean plants from the field reclaims root nodule.This root nodule is carried out surface sterilization and smash.To from root nodule, be coated on the nutrient agar flat board of nutrition by the strain separated cell, and therein it be cultivated and send back in the laboratory.
In the laboratory, make the culture of bacterial strain longer, analyze with unidirectional polyacrylamide gel electrophoresis then.Make isolated each root nodule bacterium isolate generation protein gel collection of illustrative plates from each state.Then the gel pattern that obtains from isolate is compared analysis to determine that those collection of illustrative plates are the most significant in each state two specified states.This analysis can be differentiated dominant certain gel pattern from the sample of being got.With gel pattern marshalling, and specify with interim bacterial classification number (be assigned to the gel pattern group with help with collection of illustrative plates be grouped into relevant bacterial classification number).
The bacterial strain that has identified called after IA2838 from the sample of Iowa except that a kind of other Iowa bacterial strains is as the most dominant.Except that a kind of other bacterial classifications, determine the bacterial classification of a kind of called after LA1304 like that the product again from Xi'an, Louis.This is a most representative bacterial classification in gel pattern.For these programs, two bacterial classifications all are confirmed as the most dominant natural bacterial strain.
In order to confirm the infective activity of these Rhizobium strains, carry out the seedling rearing room infective activity test of every kind of root nodule bacterium.The bacterium that comes from each bacterial strain is inoculated into single on the soybean seeds of seedling rearing room growth.The relative nitrogen fixing capacity of bacterial strain when doing the acetylene determination by reduction on the young soybean plants so that showing on being inoculated into soybean.Two kinds of bacterial strains all are found and can infect in soybean and form root nodule at the root of soybean plants, and show that reasonably (although not strengthening) acetylene reduction vigor is with indication fixed nitrogen situation.
For confirming that these bacterial classifications are dominant and competitive really in the place of therefrom isolating bacterial classification, in the condition of the field of reality, use natural dominant bacterial strain to carry out the field test, in the soybean field of Hawkeye State and Xi'an, Louis Na Zhou, under normal local soybean planting practice, between planting season, use this bacterial strain as inoculum.In order to study emulative difference, be inoculated in two fields in the zone from the two competitive natively bacterial strains that come about the bacterial classification of Hawkeye State and Xi'an, Louis Na Zhou.In the test of the field in each state two contrasts are arranged, first kind of contrast is need not do the plant of inoculation wittingly by any inoculum, second kind of contrast planting at commercial obtainable inoculum with the production of Nie Te lashing wire (Nitragin) company.The dominant natively bacterial strain in Iowa that identifies at Iowa place is 37% of total root nodule bacterium number of reclaiming from the root nodule of nonvaccinated plant.This points out that this bacterial strain that produces natively is enough to make the plant dross even without inoculation wittingly.In the occasion of commodity in use inoculum, found that the bacterium of the natural dominant bacterial strain in Iowa corresponding to test accounts for 30% of total root nodule bacterium number in the root nodule that reclaims.For the plant of the inoculum of use-testing wherein, found that 67% of root nodule bacterium number in the root nodule is the natural dominant bacterial strain in Iowa.On the contrary, found only to have in the root nodule bacterium number of root nodule 5% to be bacterium corresponding to the natural dominant bacterial strain that also is comprised in Xi'an, the Louis Na Zhou in the test inoculum.Obtain similar result at Xi'an, Louis Na Zhouchu, locate at this that Xi'an, Louis right dominant bacterial strain that day accounts for 33% in the root nodule that nonvaccinated plant has, the natural dominant bacterial strain that the plant that inoculates with Nie Te lashing wire company product provides accounts for 13% in the root nodule bacterium of its root nodule, and experimental field the natural dominant bacterial strain that plant had of inoculation accounts for 63% in its root nodule bacterium.The inoculum of test also comprises two kinds of natural dominant strainses.At the Hawkeye State place, have only 5% bacterium that comprises that bacterial strain of Xi'an, natural dominant Louis in the root nodule flora of root nodule, its result is to similar in the resulting result of the natural dominant Iowa of Xi'an, Louis Na Zhouchu bacterial strain.Thereby determined that natural dominant bacterial strain can make the plant dross effectively in the environment of field; And this bacterial strain has competitive capacity very, can compete with the dross position in the contention soybean root with existing intrinsic root nodule bacterium and other inoculums.In addition, also understood for each place in two selected geographic areas by this test; All be better than the natural dominant bacterial strain that obtains from another place in each place from these local resultant natural dominant bacterial strains.
In case confirmed the competitiveness and the dross vigor of natural dominant bacterial strain, make bacterial strain carry out mutagenesis then, to select the bacterial strain that high nitrogen fixation activity is arranged.Figure 1 shows that the schema of the method that is used for realizing this step.Used chemical mutagen is a nitrosoguanidine.Bacterium is handled with mutagenic compound, shift then, and allow to grow in the liquid medium within and shift several times.Then will be the bacterium colony of mutagenesis be coated on the agar plate to obtain one bacterium colony, it is transferred in the liquid nutrient medium grow again.The bacterium that will obtain from the bacterium colony that grows is placed on the soybean seedling, and this seedling ties up to moist aseptic soybean seedling of accompanying the surface of growing in Ti Shi (Petri) culture plate to go out and grew in the soybean seeds of bacterium three days.Soybean seedling and inoculum are placed in the serum bottle that contains aseptic vermiculite and no nitrogen nutrition liquid.On the soybean seedling and growth is being arranged in the seedling rearing room of illumination 18 days, with vapor-phase chromatography determine acetylene reduction vigor after 18 days with aseptic plastics pkt. flap.The bacterium colony that will show the mutagenesis of best nitrogen fixation activity (acetylene reduction vigor) inoculates in the other plant and tests to confirm that this vigor can recur again.
Fig. 2 and the enhancing situation that Figure 3 shows that the nitrogen fixing capacity that explanation is obtained from the bacterial classification of mutagenesis.At the mutant strain with the mutagenesis of Iowa bacterial classification K567(IA2838 of indicating shown in Fig. 2) and the comparison of the relative acetylene reduction vigor of bacterial classification IA2838, its unit makes by oneself.Figure 3 shows that the similar graphic representation of bacterial classification S258 institute enhanced nitrogen fixation activity, bacterial classification S258 is the high fixed nitrogen mutant strain with that LA1304 bacterial classification of Xi'an, natural competitive Louis.
Then mutant strain S258 and K567 are used as inoculum in the test of actual field, attempt confirms nitrogen fixation activity competitive and that increase by measuring output.Ancestors test each bacterial classification with respect to its wild-type.With the bacterial strain S258 of peat bed inoculation demonstrate output than Xi'an, Louis Na Zhou on the soybean of kind Tai Nieer (centennial) kind similarly the wild-type ancestors as Inoculant to increase by 9.9%.Utilize the peat bed of being grown in the Yi Linuoyisi state to do in the similar test on No. 82 soybean of relevant William, resulting output is the same between mutant strain and wild type strain.With the bacterial classification K567 of peat bed inoculation with also be that bacterial classification IA2838 with the peat bed inoculation compares, the Cauer in the field in Ohio and Wei Shikang star state continues according to mean yield increase by 13.4% shown on No. 79, (Corsoy) and No. 82 soybean fields of William.Use nonvaccinated field and carry out the field test of two kinds of bacterial strains and also compare with using existing commercial bacterial strain (Nie Te lashing wire), two sudden change bacterial classifications express on statistics also that the output than two kinds of contrasts has phenomenal growth.
Also may further strengthen the dross competitiveness of these competitive mutant strains.Can speed if root nodule bacterium begin the speed of process of nodulation, then the first dross bacterium has occupied root nodule thereby be better than other bacterial strains on competitiveness owing to them.Learnt that soybean root extracting solution can be used for strengthening the dross vigor of soybean nodulation bacterial isolates.This phenomenon also is effective when using by the made novel bacterial of present method.
In order to confirm this effect, soybean seeds is germinateed in transparent plastic containers in the contained soil.Behind seed germination, doing the position of mark on the container with the indication tip of a root.Culture with the survival of soybean nodulation bacterial isolates K567 makes soil inoculation then.Allow plant longer, write down the root nodule of plant root then, not only write down the root nodule number but also write down its position with respect to tip of a root mark.Control plant is then inoculated with untreated K567 bacterium, the plant of test then with before wherein the nutrient broth of overgrowth beans seedling pretreated bacterium inoculate.
Its result Fig. 4 and below table 1 in show.Fig. 4 points out that total root nodule number increases in the plant of test, and the general long higher part indication root nodule in roots of plants of root nodule forms comparatively fast.Table 1 confirms this result.Even thereby for the new competitive bacterial strain that this method produced, the pre-treatment phenomenon also is effective.
Table 1
The contrast pre-treatment
Uppermost root nodule is apart from the tip of a root-0.02cm-0.49cm
Mark (rtm) is evenly distributed
(negative sign refers to be higher than r.t.m)
Below tip of a root mark 82.4% 82.5%
Dross per-cent
Dross per-cent on lateral root 32.0% 4.9%
The root nodule 1.9 8.3 of each plant on main root
The root nodule 2.7 1.5 of each plant on lateral root
Thereby this example explanation can produce the bacterial classification of the new competitive and high fixed nitrogen of rhizobium with method of the present invention. This method has two advantages than previously known method technically. In order to select competitive bacterial strain in the field, this method not necessarily will attempt to simulate competitive any field environment when managing to select competitive rhizobium bacterium. Inoculum bacterial strain in the past is not very competitive, and selects exactly competition in the environment of simulation field The prior art of property is not very ripe. In addition, this method do not need testing crew differentiate, quantify or study the rhizobium bacterial classification itself that in the field, has competitive advantage must or favourable characteristic (can become the main tenant in the root nodule of the soybean that does to grow in the substance environment in the field or other legumes). The advantage of this method is that the rhizobium vigor that is under any circumstance taking place in the farmland has natural adaptability.
Claims (14)
1, a kind of generation is characterized in that as the method for the new bacterial strain of the root nodule bacterium of the fabaceous crop inoculum in selected geographic area the step that comprises is:
Many root nodule bacterium cultures that separation comes from this geographic area;
By being compared, culture analyzes the natural dominant root nodule bacterium of differentiating in this geographic area;
The culture of natural dominant bacterial strain is handled with mutagenic compound;
In the bacterium of mutagenesis, select the bacterium that can when leguminous plants grows, have the height nitrogen fixation activity.
2, the method for claim 1 is characterized in that the step of differentiating the natural bacterial strain of preponderating includes the step that the constitutive protein matter of the bacterium in each isolated culture is made comparisons and analyzed.
3, method as claimed in claim 2 is characterized in that discerning natural dominant bacterial strain and carries out with unidirectional polyacrylamide gel electrophoresis.
4, the method for claim 1, it is characterized in that the step that after differentiating step, further comprises for test by the field of carrying out bacterial strain and the root nodule of experiment with measuring Tanaka's plant in the rate of recovery of this bacterial classification confirm the competitiveness of the natural dominant bacterial strain that identified.
5, the method for claim 1 is characterized in that the step that also comprises is by the bacterial strain of mutagenesis being done the field test and being evaluated the competitiveness that its units increased in production that produces confirms the bacterium of mutagenesis.
6, the method for claim 1 is characterized in that nitrogen fixation activity measures by the acetylene reductive is measured.
7, the method for claim 1 is characterized in that root nodule bacterium are from by the soybean nodulation bacterium, trifolium root nodule bacteria, pea root nodule bacterium, alfalfa Phylloxera bacterium, selected bacterial classification in the bacterium group that plumage French beans root nodule bacteria and Kidney bean root nodule bacteria are formed.
8, the method for claim 1 is characterized in that root nodule bacterium are soybean nodulation bacteria cultures.
9, by the root nodule bacteria of the new bacterial strain that method produced of claim 1.
10, a kind of its feature of legume inoculation composition comprises the bacterium by the new rhizobium strains that method produced of claim 1.
11, legume inoculation thing as claimed in claim 10 is characterized in that bacterium carries out pre-treatment with leguminous plants root extraction liquid.
12, the method for the new bacterial strain of a kind of generation and the symbiotic microbial strains of crop, new bacterial strain is characterized in that as this crop inoculum in selected geographic area the step that this method comprises is:
A plurality of cultures from the bacterial strain of this treatment zone separate microorganism bacterial classification;
Be identified in the natural dominant bacterial strain in this geographic area of microbial strains with the method for comparative analysis to culture;
Natural dominant bacterial classification is handled with mutagenic compound; And
In the offspring of the culture of mutagenesis, select to make the bacterial strain of the mutagenesis that plant growth improves.
13, a kind of microorganism strains that method produced by claim 12.
14, a kind of inoculum of agriculturally useful, its feature comprises the culture by the microbial strains that method produced of claim 12.
Applications Claiming Priority (4)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| US77072785A | 1985-08-21 | 1985-08-21 | |
| US770.727 | 1985-08-29 | ||
| US85686086A | 1986-04-21 | 1986-04-21 | |
| US856.860 | 1986-04-21 |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN86105904A true CN86105904A (en) | 1987-02-25 |
Family
ID=27118349
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN198686105904A Pending CN86105904A (en) | 1985-08-29 | 1986-08-28 | Rhizobium inoculants |
Country Status (3)
| Country | Link |
|---|---|
| CN (1) | CN86105904A (en) |
| BR (1) | BR8604111A (en) |
| CA (1) | CA1335434C (en) |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102503704A (en) * | 2011-11-21 | 2012-06-20 | 山东金正大生态工程股份有限公司 | Inoculation and fertilization integrated special controlled-release fertilizer for shamrocks, preparation and application thereof |
| CN103648284A (en) * | 2011-03-31 | 2014-03-19 | 诺维信生物股份有限公司 | Competitive and effective bradyrhizobium japonicum strains |
Families Citing this family (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| FR3033978B1 (en) * | 2015-03-24 | 2017-04-07 | Jouffray Drillaud | METHOD FOR CULTIVATION ON A PLOT OF ANNUAL CULTIVATION PLANTS, ADVANTAGESALLY A CULTIVATION OF SALES OR A CULTURE OF PENSION |
-
1986
- 1986-08-19 CA CA000516285A patent/CA1335434C/en not_active Expired - Fee Related
- 1986-08-28 BR BR8604111A patent/BR8604111A/en not_active Application Discontinuation
- 1986-08-28 CN CN198686105904A patent/CN86105904A/en active Pending
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103648284A (en) * | 2011-03-31 | 2014-03-19 | 诺维信生物股份有限公司 | Competitive and effective bradyrhizobium japonicum strains |
| CN103648284B (en) * | 2011-03-31 | 2016-09-21 | 诺维信生物股份有限公司 | Competitive and effective Semen sojae atricolor gives birth to rhizobium strains slowly |
| CN102503704A (en) * | 2011-11-21 | 2012-06-20 | 山东金正大生态工程股份有限公司 | Inoculation and fertilization integrated special controlled-release fertilizer for shamrocks, preparation and application thereof |
Also Published As
| Publication number | Publication date |
|---|---|
| BR8604111A (en) | 1987-04-22 |
| CA1335434C (en) | 1995-05-02 |
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