A method for promoting nodulation and nitrogen fixation of stem mustard roots by using sesbania nitrogen-fixing rhizobium

Abstract

The present invention discloses a method for utilizing sesbania nitrogen-fixing rhizobium ( Azorhizobium caulinodans ) A method for promoting nodulation and nitrogen fixation in the root system of tumorous stem mustard. During the cultivation or planting process of tumorous stem mustard, nitrogen-fixing rhizobia of sesbania are applied to its culture medium or soil, or the roots of tumorous stem mustard are soaked with nitrogen-fixing rhizobia of sesbania. The research of the present invention shows that rhizobia can not only infect leguminous plants to establish a symbiotic relationship, but also infect tumorous stem mustard through artificial induction and promote its nodulation and nitrogen fixation. Therefore, the present invention utilizes the nitrogen-fixing ability of nodules to reduce the use of chemical nitrogen fertilizers in cultivation, alleviate the dependence on chemical nitrogen fertilizers in agricultural processes, reduce environmental pollution, and increase crop yields.

Description

Technical Field

[0001] The invention relates to the field of crop cultivation, in particular to a cultivation method of tumorous stem mustard, and more particularly to a method for promoting nodulation and nitrogen fixation of the root system of tumorous stem mustard by utilizing nitrogen-fixing rhizobium of sesbania. Background Art

[0002] Tuberous stem mustard (Brassica juncea var. tumida Tsenet Lee) belongs to the family Cruciferae, class Dicotyledons, genus Brassica. The origin of mustard cultivation in China is the cold and gloomy northern regions, and then it was moved from the northern regions to Sichuan for cultivation. The history of mustard cultivation in Sichuan has been more than 100 years, and it is commonly known as "green cabbage head". Yang Yigeng and other experts from the Chongqing Institute of Agricultural Sciences in my country have carried out systematic classification and induction of mustard. "Stem mustard" is classified as a type of mustard, and "green cabbage head" is renamed "tumorous stem mustard" (Wang Zikun. Ecological and geochemical evaluation of the Tuojiang tumorous stem mustard production area in Sichuan [D]; Chengdu University of Technology, 2012.). At present, in the cultivation process of mustard, clubroot and viral diseases have seriously restricted the production of mustard, among which clubroot is the most serious, resulting in a serious decline in the yield and quality of mustard. The plots with serious disease will no longer be suitable for mustard cultivation, which seriously threatens the sustainable development of the mustard industry (Wang Diandong, Tian Xueliang, Pan Limei. Study on the diversity of fungal communities in the rhizosphere soil of mustard fields with different incidence degrees of clubroot [J]. Northern Horticulture, 2016, 13): 115.).

[0003] Zhou Na and others used lime nitrogen as base fertilizer and microbial fertilizer as topdressing in radish planting. Animal manure and straw were applied to the soil, water was sprayed to moisten it, and a film was covered to keep it moist (Zhou Na, Tao Weilin, Lu Jingwei, et al. Symptoms and comprehensive prevention of radish root root disease [J]. Yangtze River Vegetables, 2021, 03): 53.). Planting radishes under this culture medium effectively alleviated the disease base number. At the same time, the combination of lime nitrogen and microbial fertilizer alleviated the soil environment, improved the acidification problem, optimized the soil structure, and improved the fertilizer efficiency.

[0004] Rhizobium is a Gram-negative bacillus that can invade legumes and form nodules on their roots. Rhizobium and legumes establish a symbiotic nitrogen-fixing system that can promote plant growth and increase plant yields without harming the surrounding environment of plant growth. In 1888, German scientist Bayer first discovered nodules on the roots of the non-legume plant Dioscorea paniculata; Pagan et al. proved through experiments that rhizobia can live and fix nitrogen on their own in artificial culture media. This research broke the concept that rhizobia can only complete nitrogen fixation when they rely on host plants (Huang Qunzhe, Chen Qifeng, Li Zhizhen. Prospects of biological nitrogen fixation research [J]. Science and Technology Review, 17(9901)).

[0005] Although basic experiments on inducing nodulation in non-leguminous plants have made some progress, the research is still in the basic stage. More time and energy need to be invested in research in this area before it can be systematically improved and applied in actual agricultural production. Summary of the invention

[0006] According to the characteristic that rhizobia have a certain growth-promoting effect on plant growth, the present invention optimizes the rhizobia infection system of nodular mustard (especially Fuza No. 2) by screening rhizobia through indoor limit bacteria experiments, and optimizing the concentration of rhizobia liquid, so as to induce rhizobia to invade the roots of nodular mustard and colonize, and use plant growth hormones to induce the formation of nitrogen-fixing structural nodules in the roots of nodular mustard. At the same time, the influence of rhizobia on root knot disease is explored, and the nitrogen-fixing ability of the nodules is used to reduce the use of chemical nitrogen fertilizers in cultivation, and to alleviate the problem of production reduction caused by root knot disease to a certain extent, thereby alleviating the dependence on chemical nitrogen fertilizers in the agricultural process, increasing crop yields, and reducing environmental pollution.

[0007] The present invention firstly provides a method for promoting nodulation and nitrogen fixation of the root system of tumor-bearing mustard by utilizing sesbania nitrogen-fixing rhizobium. During the culture or cultivation process of tumor-bearing mustard, sesbania nitrogen-fixing rhizobium (Azorhizobiumcaulinodans) is applied to the culture medium or soil thereof, or the roots of the tumor-bearing mustard are immersed in sesbania nitrogen-fixing rhizobium.

[0008] Preferably, the timing of applying the nitrogen-fixing rhizobium of Sesbania is during the seedling stage of the tuber mustard, or the roots of the tuber mustard seedlings are immersed in the nitrogen-fixing rhizobium of Sesbania.

[0009] In a more preferred embodiment, one, two or three inducers selected from 2,4-D, chlorsulfuron or leguminous acid are simultaneously applied when applying the nitrogen-fixing rhizobium of Sesbania sesbania. Among them, leguminous acid or 2,4-D is particularly preferred. In a specific embodiment, the concentration of the inducer is 0.5 mg / l to 2.5 mg / l, and most preferably 1 mg / l.

[0010] In another preferred embodiment, the applied sesbania nitrogen-fixing rhizobium is sesbania nitrogen-fixing rhizobium cultured to the logarithmic growth phase, and the applied concentration is 1.0×10 6 spores / mL to 1.0×10 9 spores / mL, and the most preferred is 1.0×10 8 spores / mL.

[0011] In a specific embodiment, the tumorous stem mustard is Fuza No. 2, Yong'an Chuanqi, Yong'an Xiaoye, Gaoshanqing, Zaoqing No. 2, Chengdu Pixian Zhacai, and especially Fuza No. 2.

[0012] The present invention also provides application of the above method in the cultivation of tumorous stem mustard, which is used to promote nodulation and nitrogen fixation of the root system of the tumorous stem mustard.

[0013] The method of the present invention can make rhizobia invade and colonize the roots of the nodular mustard, and further use plant growth hormones to induce the formation of nitrogen-fixing structural nodules in the roots of the nodular mustard, so that the nitrogen-fixing ability of the nodules can be used to reduce the use of chemical nitrogen fertilizers in cultivation, thereby alleviating the dependence on chemical nitrogen fertilizers in the agricultural process, increasing crop yields, and reducing environmental pollution. The present invention has great application value. BRIEF DESCRIPTION OF THE DRAWINGS

[0014] Figure 1 Distribution of rhizobia in the root tissues of six different varieties of stem tumor mustard. A-Fuza 2; B-Yong'an Xiaoye; C-Yong'an Chuanqi; D-Hanqing 2; E-Gaoshanqing; F-Chengdu Pixian Zhacai.

[0015] Figure 2 The physiological indexes of six different varieties of tumorous stem mustard are shown in Figure 1. Among them, 1-Yong'an Chuanqi; 2-Yong'an Xiaoye; 3-Fuza No. 2; 4-Hanqing No. 2; 5-Gaoshanqing; 6-Chengdu Pixian Zhacai. Different lowercase letters indicate significant differences (P < 0.05), the same below.

[0016] Figure 3 At the same time, GFP-labeled A. caulinodans colonized the roots of Fuza 2 seedlings under a fluorescence microscope.

[0017] Figure 4 The difference of physiological indexes after treatment with different concentrations of inducers. Among them, 1-2.4-D-0.5mg / l; 2-2.4-D-1mg / l; 3-2.4-D-2.5mg / l; 4-Leucowei-0.5mg / l; 5-Leucowei-1mg / l; 6-Leucowei-2.5mg / l; 7-Chlorsulfuron-0.5mg / l; 8-Chlorsulfuron-1mg / l; 9-Chlorsulfuron-2.5mg / l.

[0018] Figure 5 The difference of nodulation after different inducers and different inoculation amounts at 30 days. A-1ml 2.4-D; B-1m Doukewei; C-1ml chlorsulfuron; D-5ml 2.4-D; E-5m Doukewei; F-5ml chlorsulfuron.

[0019] Figure 6 The difference of nodulation after 60 days of treatment with different inducers. A-2.5mg / l 2,4-D; B-2.5mg / l chlorsulfuron; C-2.5mg / l leguminous; D-2.5mg / l 2,4-D; E-2.5mg / l chlorsulfuron; F-2.5mg / l leguminous.

[0020] Figure 7 Difference in the number of nodulation plants treated with different inducers at 60 days.

[0021] Figure 8 Differences in physiological indicators after 60 days of treatment with different inducers.

[0022] Fig. 9 Rhizobia in the interstitial spaces between nodules under a fluorescence microscope.

[0023] Fig.10 Nitrogenase activity in roots and soil of Brassica juncea. DETAILED DESCRIPTION

[0024] The present invention is further described below through specific implementation modes, but does not constitute a limitation of the present invention.

[0025] Wherein, the reagents used in the embodiment are as follows:

[0026] Test strains: Azorhizobium caulinodans ORS571 (A.caulinodans), provided by the Institute of Plant Protection, Hunan Academy of Agricultural Sciences (Chen Yongchao, Qi Huaiting, Wang Xiaojing, et al. Study on the growth-promoting effect of Azorhizobium caulinodans on wheat leaf tissue [J]. Journal of Northwest Botany, 2016, 36(7): 1383-1390.)

[0027] Seed materials: Fuza No. 2, Yong'an Legend, Yong'an Small Leaf, Gaoshanqing, Zaoqing No. 2, Chengdu Pixian Zhacai. Among them, Fuza No. 2, Yong'an Legend, Yong'an Small Leaf, were purchased from Fuling Seed Company, Chengdu Pixian Zhacai seeds were purchased online, Gaoshanqing and Zaoqing No. 2 are new varieties selected by the Zhacai breeding team of Yangtze Normal University and are currently undergoing variety approval.

[0028] TY liquid medium: tryptone 5g / L, yeast powder 3g / L, CaCl2·2H2O 0.88g / L, pH 7.4) is used for the activation and expansion of A.caulinodans. It is cultured at 28℃, 200r / m until the logarithmic growth phase.

[0029] Sterile agar-water solid culture medium: 5 g agar powder, 1000 mL distilled water.

[0030] PBS buffer: KH2PO4 0.24g / L, KCl 0.2g / L, NaCl 8g / L, Na2HPO4 1.44g / L, pH 7.4, used to dilute the rhizobium solution.

[0031] Data processing in the embodiment: The experimental data were sorted by Execl2010, and data statistical analysis was performed using SPSS. The root length and plant height of the stem tumor mustard infected with different varieties, treated with different concentrations of rhizobia, and treated with different inducers were tested for normality. The differences between the control group and the treatment group were compared by one-way analysis of variance, and multiple comparisons were performed using the least significant difference method (LSD). Execl was used to draw bar graphs and error bars.

[0032] Unless otherwise described in detail, the specific operation steps in the embodiments are all conventional methods.

[0033] Example 1: Study on the growth-promoting effect of rhizobia on tumorous stem mustard

[0034] 1. Seed treatment and bacterial solution preparation

[0035] Select uniform, full, and roughly the same size of stem tumor mustard seeds and place them in 2ml centrifuge tubes. Place the centrifuge tubes in a clean bench and soak the seeds in 50% NaClO for 10 minutes. Shake the centrifuge tubes during soaking to ensure that all seeds are soaked. Pour out the cleaning solution and use a pipette to absorb sterile water for rinsing and surface disinfection. Replace the rinsing solution with a pipette, and use a 1ml sterilized pipette tip. Place the surface-sterilized seeds in a tissue culture bottle, pour the high-temperature sterilized agar-water solid culture medium (hereinafter referred to as the culture medium) on the surface of the tissue culture bottle, and perform limited bacterial culture. Place it on a conventional culture rack for culture.

[0036] Activation and expansion of rhizobia: Add TY medium to a small glass bottle, add rhizobia to the TY medium under sterile conditions, seal the glass bottle with a sealing film and place it on a shaker. Set the temperature to 28°C and culture at 200r / min for 48 hours to activate it. Pipette 4ml of activated A.caulinodans solution into 200ml of TY medium with a 1ml pipette and culture on a shaker until the logarithmic growth phase (OD 600 =0.6-0.8). The activated bacterial solution and sterilized 30% glycerol were placed in a 1.5 ml centrifuge tube at a ratio of 1:1 and stored in a -80°C refrigerator for later use. A. caulinodans cultured to the logarithmic growth phase was placed in a 50 mL centrifuge tube, centrifuged at 4500 r / m for 10 min to collect the cells, diluted with PBS buffer, and the bacterial solution concentration was diluted using a hemocytometer.

[0037] 2. Experiment on infection of different varieties of tuberous mustard with rhizobia

[0038] Six varieties of mustard seeds were selected, and the surface disinfection method was the same as above. The seeds were placed in sterilized agar tissue culture bottles, 10 seeds per bottle for each variety, and 5 replicates were set. The seedlings were used for rhizobium inoculation experiments 2 days after seed germination. Seedlings with roughly the same growth status were selected, and the number of dilute rhizobia was pipetted to 1.0×10 8cfu / mL bacterial solution was added to the agar medium. The control group was inoculated with an equal amount of PBS buffer, with 5 replicates. The culture conditions were the same as above, and the growth of stem tumor mustard was investigated 9 days after inoculation. The roots of the seedlings were rinsed with tap water, then rinsed three times with sterile water, and placed on a black table to take pictures after natural air drying. The root length and plant height were then measured with a ruler, and representative seedlings were taken for fluorescence microscopy observation.

[0039] 3. Experimental results

[0040] Distribution of nitrogen-fixing rhizobia in the root tissue of Sesbania sesbania seedlings Figure 1 As shown. Nine days after inoculation, rhizobia were detected by fluorescence microscopy to be colonized in the roots of all varieties of mustard. The colonization sites of rhizobia in the roots of different varieties of mustard were roughly the same, mostly colonizing the epidermal cells and intercellular spaces of the seedling roots, but the specific distribution numbers were different. Among them, the largest number of rhizobia was detected in the root tissue of Fuza No. 2, followed by Yong'an Chuanqi and Yong'an Xiaoye, and the least number of rhizobia was Hanqing No. 2. Bright GFP fluorescence was observed in the root tissue of Fuza No. 2 ( Figure 1 In the middle (A), the GFP marker detected is significantly more than that of other mustard varieties. The rhizobia colonized the roots of mustard in a scattered and dotted manner. The experiment proved that rhizobia can also exist in the internal tissue of the nodules of non-leguminous mustard.

[0041] Example 2 Infection experiment of different concentrations of rhizobia against tumorous stem mustard

[0042] The surface of Fuza No. 2 seeds was disinfected in the same way as above, and placed on agar-water solid medium for capping bacteria culture. When the seeds germinated to the two-leaf and one-heart stage, the ungerminated seeds were removed with tweezers, leaving seedlings with roughly the same growth status. The rhizobium solution in the logarithmic growth period was placed in a 50ml centrifuge tube, centrifuged at 4500r / m for 10min to collect the bacteria, and diluted with PBS buffer to 1.0×10 9 cfu / mL, 1.0×10 8 cfu / mL, 1.0×10 7 cfu / mL, 1.0×10 6 cfu / mL four different concentrations were prepared. 1 ml of rhizobium solution of different concentrations was respectively aspirated onto the surface of the culture medium, with 4 replicates for each concentration, 10 grains per bottle, and an equal amount of PBS was inoculated for parallel control, with 4 replicates. After 9 days of inoculation, the growth of the tumorous stem mustard seedlings was investigated and the root length and plant height were measured with a ruler and the data were tested for normality. The root length and plant height data were in accordance with the normal distribution.

[0043] The results of physiological index tests showed that A. Caulinodans had different effects on root length and plant height of different stem tumor mustard varieties. According to the LSD test, the root length and plant height of Fuza 2 seedlings inoculated with fungi were different from those of the control ( Figure 2 ), the root length and plant height of Fuza 2 and Hanqing 2 were significantly higher than those of the control group (P<0.05), the root length of Yongan Xiaoye was not significantly different from that of the control group (P>0.05), but the plant height was significantly different (P<0.05), the root length of Yongan Chuanqi was significantly different from that of the uninoculated group (P<0.05), but the plant height was not significantly different (P>0.05), the root length of Gaoshanqing and Chengdu Pixian Zhacai was significantly different (P<0.05), but the plant height was not significantly different (P>0.05).

[0044] Compared with the control group, the root length of Yong'an Xiaoye increased by 4.5%, and the plant height increased by 3%. Compared with the control group, the root length of Yong'an Chuanqi increased by 4.1%, and the plant height increased by 4.8%. Compared with the control group, the root length of Hanqing No. 2 increased by 3.2%, and the plant height increased by 2.5%. Compared with the control group, the root length of Chengdu Pixian Zhacai increased by 8.6%, and the plant height increased by 11%. Compared with the control group, the root length of Fuza No. 2 increased by 11.55%, and the plant height of Fuza No. 2 increased by 12.06%. Compared with the control group, the root length of Gaoshanqing increased by 5.5%, and the plant height increased by 6.8%. Among them, the root length and plant height of Fuza No. 2 had the most obvious growth-promoting effect after rhizobium infection, and the increase in root length and plant height of Fuza No. 2 was twice that of Gaoshanqing.

[0045] In summary, appropriate concentrations of rhizobia have a growth-promoting effect on the growth of different varieties of tumorous stem mustard. Among them, the root length and plant height of the inoculated seedlings of Fuza 2 were significantly different from those of the control group. Fuza 2, as a commonly used variety for tumorous stem mustard cultivation, has a good growth state. Therefore, Fuza 2 was selected to further study the molecular mechanism of the interaction between A. caulinodans and tumorous stem mustard.

[0046] Example 3 Observation of the colonization of rhizobia in the roots of Brassica juncea

[0047] Under indoor conditions, the surface of plump seeds of Fuza No. 2 was disinfected in the same way as above. They were placed on agar-water solid medium for cap bacteria culture. When they germinated to the two-leaf and one-heart stage, 1 mL of 1.0×10 8 cfu / mL PBS diluted bacterial solution was added to the surface of agar-water solid culture medium to inoculate the stem mustard seedlings. After inoculation, the tissue culture bottle was sealed with sealing film to avoid contamination. A conventional intelligent culture rack was placed for limited bacterial culture. A time gradient was set at 3d, 6d, 9d, 12d, 15d, 18d, and 21d after inoculation, and the colonization of GFP-labeled A.caulinodans in the roots of the stem mustard seedlings was regularly detected with a fluorescence microscope.

[0048] Select representative tumorous mustard seedlings under each time period, prepare 3-4% agarose aqueous solution and put it into a 100ml conical flask, heat the agarose in the middle of the microwave oven to melt, pour the melted agarose into a centrifuge tube with a diameter of 2cm, and embed the materials to be sliced ​​in the agarose, and put it on ice to solidify. After the blade is surface disinfected with 75% alcohol, use the blade to cut the roots of the test seedlings into thin slices by hand, put them on a slide, and absorb an appropriate amount of sterilized 50% glycerol and place them on the slide. Wipe the blade with 75% alcohol before each use, and sterilize the blade with an alcohol lamp to avoid cross contamination.

[0049] The finished slices are fixed on the slice holder, and the low-power microscope is used to observe the tissue under the white light field of view, and then the coarse and fine focus knobs are adjusted to the appropriate observation magnification, and the white light source is switched to green fluorescence to observe the green fluorescence signal on the tissue surface. After the fluorescent colonization tissue site is determined, take a photo under the green fluorescence light source, and then return to the white light source to take a photo. The photos under the two light sources are printed into a composite image so that the fluorescence distribution is presented on the sample tissue under the white light source, and finally the image is output and saved.

[0050] The fluorescence detection results showed that ( Figure 3 ), rhizobia colonized the root tissue of seedlings 3 days after infecting tuber mustard, and the colonization of rhizobia increased over time. At 15 days after inoculation, the colonization of rhizobia had multiplied in large numbers on the roots of seedlings and developed into a certain range of population density locally. A. caulinodans can not only infect non-leguminous plants, but also colonize in the intercellular spaces and cells of the epidermis and cortex of plant roots to form an ecological niche. Fluorescence microscopy showed that the colonization pattern of rhizobia infecting the roots of non-leguminous plants is roughly similar to that of rhizobia invading leguminous plants. The way of invading and colonizing plant root tissues is "crack entry", and over time, rhizobia can form a certain range of population density on the roots of tuber mustard.

[0051] Example 4 Study on the Nodulation of Tuber Mustard Induced by Rhizobium Nodulation Inducer

[0052] 1. Effects of different concentrations of rhizobium nodulation inducers on root nodulation of Brassica juncea

[0053] The three inducers, 2,4-D, chlorsulfuron and benzimidazole, were prepared into high-concentration mother solutions and placed in a refrigerator at 4 degrees for later use. The three concentration gradient dilutions were 0.5 mg / l, 1 mg / l, and 2.5 mg / l, respectively. At the same time, the A. caulinodans in the logarithmic growth phase was diluted with PBS buffer to a concentration of 1.0 × 10 8 cfu / mL for future use.

[0054] The surface disinfection method of seeds was the same as above. Filter paper was placed in a culture dish for high temperature sterilization. The filter paper was soaked in sterile water to keep it moist. The surface-sterilized stem tumor mustard Fuza No. 2 seeds were placed on the filter paper for germination. The sealing film was pasted on the culture dish for limited bacterial culture. Ten seeds were treated with each of the three inducers, with 5 replicates. After germination to the two-leaf and one-heart stage, different concentrations of inducers were added to each treatment, and then an equal amount of 1 mL of 1.0×10 8 cfu / mL A.caulinodans bacterial solution, culture conditions are the same as above.

[0055] After 30 days of treatment with different concentrations of inducers, 30 seedlings in each treatment group were investigated to analyze the effects of different inducers combined with A. caulinodans on the growth physiology of tumorous mustard. The results showed that the root length, plant height, leaf length, and root and leaf fresh weight of tumorous mustard were significantly different ( Figure 4 ). Among them, the 1mg / l concentration of benzimidazole had the greatest effect on the physiological growth of stem tumor mustard. The average leaf length of 2,4-D at 0.5mg / l concentration was 15.35cm, the average root length was 10.48cm, the average plant height was 18.95cm, the average leaf weight was 2.668g, and the average root weight was 0.174cm. The average leaf length of 2,4-D at 1mg / l concentration was 16.74cm, the root length was 11.52cm, the average plant height was 21.14cm, the average leaf weight was 1.32g, and the average root weight was 0.3 7g, at 2.5mg / l concentration, the average leaf length of 2,4-D was 15.49cm, the average root length was 11.8cm, the average plant height was 22.51cm, the average leaf weight was 1.227g, and the average root weight was 0.189g. At 0.5mg / l concentration, the average leaf length of Doukewei was 14.5cm, the average root length was 9.23cm, the average plant height was 24.3cm, the average leaf weight was 3.01g, and the root weight was 0.46g.

[0056] The average plant height of 1mg / l bean curd was 25.4cm, the average leaf length was 16.57cm, the average root length was 12.34cm, and the average root weight was 0.53g. The average leaf length of 2.5mg / l bean curd was 14cm, the average root length was 8.75cm, the average plant height was 21.65cm, the average leaf weight was 3.401g, and the average root weight was 0.44g. The average leaf length of 0.5mg / l chlorsulfuron was 14.45cm, the average root length was 8.86cm, and the average plant height was 21.65cm, the average leaf weight was 0.44g, and the average root weight was 0.44g. At the concentration of 1mg / l, the average leaf length of chlorsulfuron was 11.75cm, the average root length was 15.44cm, the average plant height was 22.35cm, the average leaf weight was 0.504g, and the average root weight was 0.455g. At the concentration of 2.5mg / l, the average leaf length of chlorsulfuron was 14.94cm, the average root length was 12.13cm, the average plant height was 25.92cm, the average leaf weight was 7.23g, and the average root weight was 0.63g.

[0057] In summary, the average plant height of 1 mg / l of Legume was higher than the seedling height under the three concentrations of 2,4-D and the two concentrations of 0.5 mg / l and 1 mg / l of chlorsulfuron, but lower than the plant height under the treatment of 2.5 mg / l of chlorsulfuron. The leaf length of Legume at 1 mg / l was 16.57 cm, which was higher than the leaf length under the three concentrations of 2,4-D and chlorsulfuron. The average root weight of Legume at 1 mg / l was 0.53 g, which was not much different from the root weight of 2.5 mg / l of chlorsulfuron, but higher than the root weight under other treatments. In summary, three concentrations of nodulation inducers were used to induce tumorous stem mustard, and 1 mg / l of Legume did not show a significant inhibitory effect on the physiological growth of tumorous stem mustard. Under the treatment of 1 mg / l of Legume, the growth state of Fuza No. 2 seedlings was the best.

[0058] 2. Effects of different doses of rhizobium nodulation inducers on root nodulation of Brassica juncea

[0059] Seed disinfection method is the same as above, limited bacteria culture. Select three inducers with a dilution concentration of 2.5 mg / l and a dilution of 1.0×10 8 cfu / mL A.caulinodans suspension was set aside, and when the seeds were cultured to two leaves and one heart, they were moved to flower pots. Three inducers with an inoculum volume of 1ml and 5ml of 2.5mg / l were added around the seedlings by injection, and an equal amount of rhizobium solution was added at the same time. Ten plants were treated in each treatment, with 4 replicates. After 30d and 60d, the differences in nodulation of stem mustard under each treatment were observed.

[0060] The nodulation of stem mustard treated with different doses of rhizobium nodulation inducer was investigated 30 days after inoculation. Figure 5). The results of the investigation showed that the three inducers did not induce nodule formation in the mustard at the concentration of 2.5mg / l and 1ml, but the three inducers at the concentration of 2.5mg / l and the dosage of 5ml all induced nodule formation in the roots of the mustard. This phenomenon shows that the amount of inducer used is an important factor affecting the nodulation of mustard. When 5ml of 2,4-D and rhizobia were used simultaneously, the nodulation effect of mustard was the most obvious and the most nodules were formed, followed by leguminous and chlorsulfuron. The nodules formed by 2,4-D and chlorsulfuron were roughly the same in morphology, both of which were semi-spherical, while the nodules formed by leguminous were semi-spherical and short rod-shaped. Under the same culture conditions, the control group of mustard, which was cultured with rhizobia only without the addition of inducers, did not induce nodules to form on the roots of mustard, although rhizobia colonized the root tissue.

[0061] After 60 days of treatment, the nodule-like morphology of the tumorous stem mustard was investigated. Figure 6 The experiment used three inducers with a final concentration of 2.5 mg / l and a dose of 5 ml. When the stem tumor mustard germinated to the two-leaf and one-heart stage, 5 ml of the inducer and 1.0×10 8 cfu / mL concentration of rhizobium liquid, 60 days later, 30 stem mustard plants under three treatments were investigated. The investigation results showed that under the three different inducer treatments, the nodulation effect was still the best when the 2,4-D concentration was 2.5mg / l and the dosage was 5ml. The nodulation number ( Figure 7 ), the nodulation rate was the highest, reaching 93%, the nodulation rate of 56.7% for doukowei, and the lowest nodulation rate of 36% for chlorsulfuron. The nodules formed under 5ml of 2,4-D treatment were distributed in the main root and lateral roots of stem nodule mustard, and the number of nodules was large. Most of the nodules formed were semi-spherical, which was roughly similar to the nodules formed by wheat and most legumes in the Gramineae family. The nitrogen-fixing nodules induced by doukowei had rich morphology, not only semi-spherical nodules formed under 2,4-D treatment, but also some nodules with short rod shape. The nodules formed under chlorsulfuron treatment were similar to the nodules induced by 2,4-D and were semi-spherical.

[0062] In summary, the effect of chlorsulfuron on inducing nodulation of tumor-bearing mustard is lower than that of 2,4-D and leucovorin. 2,4-D has the best effect on inducing nodulation of tumor-bearing mustard and can be used as an inducer in inducing nodulation production of tumor-bearing mustard.

[0063] Among them, nodulation rate = (number of nodulated plants / number of investigated plants) × 100%

[0064] 3. Analysis of differences in physiological indicators of tumorous stem mustard treated with different doses of inducers

[0065] The differences in physiological indexes of 30 tumorous stem mustard plants after treatment with different doses of inducers for 60 days were investigated. Figure 8). The fresh root weight of stem mustard treated with 2,4-D at a concentration of 2.5 mg / l and a dose of 5 ml and rhizobium solution was the largest, with an average of 1.47 g. Its root weight was significantly different from that treated with leguminous and chlorsulfuron, 5% lower than that of the control group, and 12% and 11% higher than that of chlorsulfuron and leguminous. According to the least significant difference test, 2,4-D was not significantly different from the control group (P>0.05), but was significantly different from chlorsulfuron and leguminous (P<0.05). In summary, the use of the three inducers and bacteria had a certain effect on the growth of stem mustard compared with the control group. Among them, the root weight of 2,4-D was not significantly different from that of the control group, indicating that 2,4-D had no significant inhibitory effect on the growth of stem mustard, while chlorsulfuron and leguminous had a significant inhibitory effect on the root weight of stem mustard. Therefore, 2,4-D, which had the best nodulation effect and had less effect on the growth of stem mustard, was selected as the inducer for the later experiments.

[0066] 4. Observation of root nodules by fluorescence microscope

[0067] The nodular Brassica juncea seedlings were taken out of the soil, rinsed with tap water, and then rinsed with sterile water to thoroughly remove the impurities on the roots, and then absorbed the water with absorbent paper or dried naturally. Representative nodular Brassica juncea roots were selected for fluorescence microscopy observation.

[0068] After nodules were formed on the roots of the mustard, the mustard seedlings that formed nodules under the treatment of 2,4-D concentration of 2.5 mg / l and dosage of 5 ml were taken to detect the colonization of A. caulinodans in the nodules. The roots of the mustard seedlings were cultured under 2,4-D and rhizobia infection for 30 days, and nodules were formed on the roots of the mustard. Through fluorescence microscopy, it was found that rhizobia could not only colonize in the natural openings of the roots of the mustard and the intercellular spaces of the root system, but also enter the intercellular spaces of the nodules and proliferate in large numbers in the nodule tissues (such as Fig. 9 ).

[0069] Example 5 Determination of nitrogenase activity in roots and soil of nodulated seedlings

[0070] Seed surface disinfection method is the same as above. When the seeds grow to the two-leaf and one-heart stage, inoculate 5 ml of 2,4-D diluted to 2.5 mg / l and 1.0×10 8 cfu / mL concentration of rhizobium solution. Under the same culture conditions, the seedlings were inoculated with 5 ml of PBS as a control, and the nodulation was observed after 30 days. After nodules were formed on the roots of the seedlings in the investigated treatment group, the nitrogenase activity was determined.

[0071] The acetylene reduction method was used to determine the nitrogenase activity of nodulated roots and culture soil. The roots of the tumorous stem mustard were rinsed, and the water was absorbed with absorbent paper and placed in a 100 ml serum bottle, which was sealed with a rubber stopper. The glass tube and the rubber tube were connected to the perforated rubber stopper and sealed to form a sealed space in the serum bottle. The air was extracted from the syringe, and acetylene gas was extracted from the gas storage bag and injected into the wide-mouth bottle for reaction for 1 hour. The various mixed gases after the reaction were transferred to an empty bottle, and the nitrogenase activity of the roots of the tumorous stem mustard was determined by the external standard method using a gas chromatograph analyzer.

[0072] Take 10g of the culture soil from the flower pot of Brassica juncea and place it in a 100ml serum bottle, add 2ml of 0.1mol / l glucose, shake evenly, cover with a rubber stopper and an aluminum cap to seal, so that a closed space is formed in the serum bottle. Use a syringe to extract acetylene gas from the bottle and culture at 280℃ for 48h. After the culture is completed, use a 1ml syringe to draw a gas sample from the bottle, and determine the nitrogenase activity of the culture soil in the same way as above.

[0073] Depend on Fig.10 It can be seen that the nitrogenase activity of the stem tumor mustard inoculated group and the control group was significantly different (p<0.05), the nitrogenase activity of the root nodules was 30.21IU / g, and the control group was 28.57IU / g; the soil of the cultivated stem tumor mustard was also tested, and the difference between the control group and the treated group was significant (p<0.05), the nitrogenase activity of the treated group was 31.73IU / g, and the control group was 29.7IU / g. In summary, although the nitrogenase activity of the treated group was higher than that of the control group, the control group that was not inoculated with rhizobia showed a certain nitrogenase activity as did the treated group, which may be because during the inoculation process, the rhizobia carried in the air migrated to the control group, which also affected it to a certain extent.

[0074] Among them, nitrogenase activity per unit nodule weight SNA = (reduced ethylene amount / nodule dry weight) × time.

Claims

1. A method for promoting nodulation and nitrogen fixation of the root system of Tuber mustard using nitrogen-fixing rhizobia of Sesbania sesbania, characterized in that: During the cultivation or planting of tumorous stem mustard, nitrogen-fixing rhizobium sesbania ( Azorhizobium caulinodans ), or soak the roots of tuber mustard with sesbania nitrogen-fixing rhizobia, and then continue to culture or cultivate; The tumorous stem mustard is Fuza No. 2; The timing of applying the nitrogen-fixing rhizobium of sesbania is during the seedling stage of the mustard, or the roots of the mustard seedlings are immersed in the nitrogen-fixing rhizobium of sesbania; and the 2,4-D inducer is applied simultaneously when applying the nitrogen-fixing rhizobium of sesbania, and the concentration of the inducer is 0.5 mg / l to 2.5 mg / l.

2. The method according to claim 1, characterized in that The concentration of the inducer was 1 mg / l.

3. The method according to claim 1, characterized in that The applied nitrogen-fixing rhizobium of Sesbania sesbania was cultivated to the logarithmic growth phase, and the applied concentration was 1.0×10 6 spores / mL to 1.0×10 9 spores / mL.

4. Use of the method according to any one of claims 1 to 3 in the cultivation of tumorous stem mustard.

5. The use according to claim 4, characterized in that It is used to promote nodulation and nitrogen fixation in the roots of tumorous stem mustard.

6. The use according to claim 4, characterized in that The roots of the mustard seedlings were soaked in sesbania nitrogen-fixing rhizobia for 20-60 minutes.

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