WO2021057446A1 - 一种促进刺槐结瘤固氮的混合菌及其应用 - Google Patents

一种促进刺槐结瘤固氮的混合菌及其应用 Download PDF

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WO2021057446A1
WO2021057446A1 PCT/CN2020/113434 CN2020113434W WO2021057446A1 WO 2021057446 A1 WO2021057446 A1 WO 2021057446A1 CN 2020113434 W CN2020113434 W CN 2020113434W WO 2021057446 A1 WO2021057446 A1 WO 2021057446A1
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liquid
bacillus
mixed
microbacterium
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庄家尧
刘超
王晓雪
郑佳欣
田琨
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南京林业大学
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    • C12N1/20—Bacteria; Culture media therefor

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  • the invention belongs to the technical field of microorganisms, and specifically relates to a mixed bacteria that promotes nodulation and nitrogen fixation of Robinia pseudoacacia and its application.
  • Guest soil spraying technology is a greening technology in which a mixture of soil growth substrate materials and plant seeds are sprayed evenly and at high pressure on the rocky soil slope.
  • the growth substrate material mixture of this technology is prepared with well-selected soil bacteria, which is mainly a greening technology developed for hard slopes such as rocks and for plants to create a suitable growth environment on hard slopes.
  • the selected soil bacteria play two roles: First, it can respond to changes in soil ecological mechanism and environmental stress, accelerate the erosion of rock mass, and effectively improve the rock wall and spraying. Matrix interface fusion; the second is to promote the growth and development of plants to ensure the supply of nutrients such as carbon and nitrogen in the stress environment.
  • soil microorganisms convert inert nitrogen in the air into ionic nitrogen that can be directly absorbed by vegetation to ensure the nitrogen nutrition of vegetation; at the same time, the stomatal conductance of vegetation treated by microorganisms with a growth-promoting effect increases High, can accelerate the gas exchange of plant cells, and increase the concentration of CO 2 between cells, so that the CO 2 required for photosynthesis is sufficient, which further increases the photosynthetic rate of plants and promotes plant photosynthesis; in addition, microorganisms play a role in their life activities. In the process, it can decompose difficult minerals in the soil to degrade inorganic and organic pollutants. These all ensure the supply of nutrients required by the vegetation in the microenvironment and the normal progress of the life activities of the vegetation. Therefore, the obvious and targeted screening of growth-promoting bacteria for specific vegetation is one of the keys to the wide application of the spraying and greening technology. At present, there are few reports on the selection of suitable legume vegetation nodule-promoting strains for different vegetations in China.
  • the technical problem to be solved by the present invention is to screen suitable leguminous vegetation nodule-promoting strains, and provide a mixed bacteria that promotes nodulation and nitrogen fixation of Robinia pseudoacacia, which is a useful tool for spraying matrix growth-promoting bacteria. Establish and provide strain support.
  • Another object of the present invention is to provide the application of the above-mentioned mixed bacteria in promoting nodulation and nitrogen fixation of Robinia pseudoacacia. It can produce a synergistic additive effect, provide a high level of nitrogen, and promote the nodulation and nitrogen fixation of Robinia pseudoacacia.
  • Another object of the present invention is to provide the application of the above mixed bacteria in promoting the growth of Robinia pseudoacacia. Each growth index has a certain increase, and the growth is good.
  • a mixed bacteria that promotes nodulation and nitrogen fixation of Robinia pseudoacacia consisting of Kocuria sp. X-22, Microbacterium sp. X-26, and Bacillus sp. X-28; among them,
  • Microbacterium sp. X-26 deposited in the China Type Culture Collection, preservation date: April 8, 2019, preservation number CCTCC No: M 2019238, preservation address: Wuhan University, Wuhan, China;
  • Bacillus (Bacillus sp.) X-28 deposited at the China Type Culture Collection, preservation date: April 8, 2019, preservation number CCTCC No: M 2019239, preservation address: Wuhan University, Wuhan, China.
  • the mixed bacteria are respectively prepared into fermentation broths, and the respective fermentation broths are diluted and mixed and then directly watered on the rhizosphere soil of the black locust seedlings.
  • the preparation method of the fermentation broth is as follows:
  • step 4) Before use, the fermentation broth obtained in step 3) is diluted with sterile water, and then mixed and applied in equal volumes.
  • the liquid medium is 10 g peptone, 3 g yeast powder, 5 g sodium chloride, 1000 mL sterile water, and pH 5.6.
  • the mixed bacteria are respectively prepared into fermentation broths, and the respective fermentation broths are diluted and mixed and then directly watered on the rhizosphere soil of the black locust seedlings.
  • the preparation method of the fermentation broth is as follows:
  • step 4) Before use, the fermentation broth obtained in step 3) is diluted with sterile water, and then mixed and applied in equal volumes.
  • the liquid medium is 10 g peptone, 3 g yeast powder, 5 g sodium chloride, 1000 mL sterile water, and pH 5.6.
  • the present invention screens and identifies Kukella X-22 strain, Microbacterium X-26 strain, and Bacillus X-28 strain from soil, and activates these three strains separately And shake culture to obtain the fermentation bacteria liquid, the fermentation bacteria liquid is diluted and mixed in equal proportions to obtain the application bacteria liquid, which can be applied to the planted Robinia pseudoacacia seedlings, which can produce synergistic and superimposing effects, and significantly increase the nodulation rate of the Robinia pseudoacacia and the level of symbiotic nitrogen fixation. , To promote the photosynthesis of Robinia pseudoacacia, and provide a new choice for spraying substrate growth-promoting bacteria.
  • Figure 1 is a diagram of the colonies of Kukella X-22, Microbacterium X-26 and Bacillus X-28 on a nutrient agar solid medium; in the figure, A: Kukella X-22; B: Microbacterium X-26; C: Bacillus X-28.
  • the cultivation of black locust seedlings was carried out in a greenhouse at the Baima Teaching Base of Nanjing Forestry University, with an air humidity of 65%; a CO 2 concentration of 450 ppm; and a maximum photosynthetically active radiation of 1850 ⁇ mol/(m 2 ⁇ s). Ensure that each potted plant receives the same light intensity and time every day. In order to prevent the roots of Robinia pseudoacacia from being rotted due to too frequent watering, watering is carried out by weighing method every time water is added to ensure that the soil water content of each pot reaches 100% of the field water holding capacity.
  • Accelerating germination adopt stratification accelerating germination method. Mix the swollen seeds with 3 times the amount of wet sand (you can loosen it by hand). In order to ensure the humidity of the sand, the surface is covered with a plastic wrap with some holes, and a thermometer is inserted and placed in a dark place. Induce germination for 3 to 4 days and keep the temperature at about 20°C.
  • step 4) Before use, the fermentation broth obtained in step 3) is diluted 100 times with sterile water, then mixed in equal volume and used.
  • a single-bacteria control group (X-22, X-26, and X-28 fermentation bacteria liquid dilutions take 60 mL respectively), a blank control group (sterile liquid fermentation medium), a mixed bacteria group (three kinds of fermentation bacteria liquids) Take 20mL each of the dilutions, a total of 60mL bacterial solution), and add them to the planted Robinia pseudoacacia seedlings by pouring in the rhizosphere soil (the first 1-4 weeks is the thinning period, no bacteria are applied, and the next 5-16 weeks is the bacteria application Culture observation period). Three parallels are set for each treatment.
  • the activated bacteria liquid is mixed and added to the surrounding of the planted locust seedlings.
  • the ground diameter is used to indicate the size of the trees. Good growing plants generally have a larger ground diameter. It can be seen from Table 3 that the ground diameter of the black locust seedlings treated with X-22 is lower than that of the sterile seedlings, and the other treatments are all higher than the sterile seedlings. The ground diameter of the seedlings treated with mixed bacteria was significantly higher than that of the sterile seedlings, an increase of 36.84% (P ⁇ 0.05).
  • Seedling height is one of the most basic indicators of plant morphology, which can directly reflect the growth status of vegetation. In general, plants that grow well have relatively high seedling heights. It can be seen from Table 3 that the height of the seedlings treated with various soil bacteria was higher than that of the aseptic seedlings, with an average seedling height of 44.58 cm and an average increase of 11.34%. Among them, the height of the seedlings treated with mixed bacteria was significantly higher than that of sterile seedlings and increased by 29.7% (P ⁇ 0.05).
  • Leaf area is one of the indicators most closely related to yield. The increase in plant yield can be directly reflected by leaf area. In general, a proper size of leaf area can make full use of light conditions without affecting photosynthesis. It can be seen from Table 3 that the leaf area of the seedlings treated with X-22 and X-28 was lower than that of the sterile seedlings. The average leaf area is 45.19mm 2 and the average increase is 3.81%. The leaf area of seedlings treated with mixed bacteria was significantly higher than that of sterile seedlings, increasing by 21.71% (P ⁇ 0.05).

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Abstract

一种促进刺槐结瘤固氮的混合菌及其应用，属于微生物技术领域。所述混合菌由库克氏菌属(Kocuria sp.)X-22，微杆菌属(Microbacterium sp.)X-26，芽孢杆菌(Bacillus sp.)X-28组成，各菌均已保藏于中国典型培养物保藏中心，保藏编号分别为：CCTCC No:M 2019237；CCTCC No:M 2019238；CCTCC No:M 2019239。将混合菌添加到刺槐幼苗周围，与单菌处理组和无菌处理组相比，能够产生增效的叠加效果，明显提高刺槐的结瘤率和共生固氮的水平，促进刺槐的光合作用。

Description

一种促进刺槐结瘤固氮的混合菌及其应用 技术领域
本发明属于微生物技术领域，具体涉及一种促进刺槐结瘤固氮的混合菌及其应用。
背景技术
客土喷播技术是将土壤生长基质材料的混合物和植物种子均匀、高压喷播在岩土坡面上的绿化技术。该技术的生长基质材料混合物中配制有筛选好的土壤菌，主要是针对岩石等硬质边坡以及为植物能够在硬质边坡营造适宜生长环境研发的一种绿化技术。所筛选的土壤菌作为该技术生长基质材料的重要成分之一发挥着两个作用：一是能对土壤生态机制变化和环境胁迫做出反应，加速岩体的侵蚀，有效提高岩壁与喷播基质界面融合性；二是促进植物的生长发育，保证植物在胁迫环境中碳素、氮素等营养的供应。然而在实际工程中，这一技术的利用受到很多的限制，一方面是喷播基质难以长久在岩面维持，另一方面针对喷播树种能够起到促生作用的土壤微生物的发现很少。
土壤微生物在生命活动中将空气中的惰性氮素转化为植被可直接吸收的离子态氮素，保证植被的氮素营养；同时，经具有促生作用的微生物处理过的植被的气孔导度升高，能够加快植物细胞的气体交换，且胞间CO 2浓度增加，使光合作用所需的CO 2充备，进而更提高了植物的光合速率，促进植物光合作用；此外，微生物在其生命活动过程中可分解土壤中难容的矿物降解无机和有机污染物。这些都保证了植被在微环境中所需各营养成分的供应以及植被各生命活动的正常进行。因此明显的、有针对性的筛选特定植被的促生菌是客土喷播绿化技术能够广泛应用的关键之一。目前，国内针对不同植被，筛选适宜的豆科植被根瘤促生菌株鲜有报道。
发明内容
针对现有技术中存在的不足，本发明所要解决的技术问题是筛选适宜的豆科植被根瘤促生菌株，提供一种促进刺槐结瘤固氮的混合菌，为喷播基质促生菌菌库的建立提供菌种支持。本发明的另一目的是提供上述混合菌在促进刺槐结瘤固氮中的应用。能够产生增效的叠加效果，提供高水平的氮素，促进刺槐结瘤固氮。 本发明的再一目的是提供上述混合菌在促进刺槐生长中的应用。各生长指标均有一定的增长，长势良好。
技术方案：为了解决上述技术问题，本发明采用的技术方案为：
一种促进刺槐结瘤固氮的混合菌，由库克氏菌属(Kocuria sp.)X-22，微杆菌属(Microbacterium sp.)X-26，芽孢杆菌(Bacillus sp.)X-28组成；其中，
库克氏菌属(Kocuria sp.)X-22，保藏于中国典型培养物保藏中心，保藏日期：2019年4月8日，保藏编号CCTCC No:M 2019237，保藏地址：中国武汉武汉大学；
微杆菌属(Microbacterium sp.)X-26，保藏于中国典型培养物保藏中心，保藏日期：2019年4月8日，保藏编号CCTCC No:M 2019238，保藏地址：中国武汉武汉大学；
芽孢杆菌(Bacillus sp.)X-28，保藏于中国典型培养物保藏中心，保藏日期：2019年4月8日，保藏编号CCTCC No:M 2019239，保藏地址：中国武汉武汉大学。
混合菌在促进刺槐结瘤固氮中的应用。
所述的应用，将混合菌分别制备出发酵菌液，将各发酵液稀释并混合后直接在刺槐幼苗的根际土壤浇施。
所述的应用，发酵菌液的制备方法为：
1)取库克氏菌属X-22、微杆菌属X-26、芽孢杆菌X-28菌株，分别在营养琼脂固体培养基上，35℃活化24h；
2)将活化后的微杆菌属X-26、芽孢杆菌X-28菌株用接种环挑取一环菌泥分别加入到LB液体培养基中，库克氏菌属X-22接入到NA液体培养基中，35℃，200r/min恒温震荡24h制备种子液；
3)按3％的接种量取种子液接种于液体培养基，35℃，200r/min摇床振荡培养36h，得到发酵菌液；
4)使用前，用无菌水将步骤3)得到的发酵菌液稀释后，等体积混合施用。
步骤3)中，液体培养基为蛋白胨10g，酵母粉3g，氯化钠5g，无菌水1000mL，pH值5.6。
混合菌在促进刺槐生长中的应用。
所述的应用，将混合菌分别制备出发酵菌液，将各发酵液稀释并混合后直接在刺槐幼苗的根际土壤浇施。
所述的应用，发酵菌液的制备方法为：
1)取库克氏菌属X-22、微杆菌属X-26、芽孢杆菌X-28菌株，分别在营养琼脂固体培养基上，35℃活化24h；
2)将活化后的微杆菌属X-26、芽孢杆菌X-28菌株用接种环挑取一环菌泥分别加入到LB液体培养基中，库克氏菌属X-22接入到NA液体培养基中，35℃，200r/min恒温震荡24h制备种子液；
3)按3％的接种量取种子液接种于液体培养基，35℃，200r/min摇床振荡培养36h，得到发酵菌液；
4)使用前，用无菌水将步骤3)得到的发酵菌液稀释后，等体积混合施用。
步骤3)中，液体培养基为蛋白胨10g，酵母粉3g，氯化钠5g，无菌水1000mL，pH值5.6。
有益效果：与现有技术相比，本发明从土壤中筛选鉴定了库克氏菌属X-22菌株、微杆菌属X-26菌株、芽孢杆菌X-28菌株，将这3种菌株分别活化并振荡培养得到发酵菌液，将发酵菌液稀释后等比例混合得到应用菌液，施用于栽植好的刺槐幼苗，能够产生增效的叠加效果，明显提高刺槐的结瘤率和共生固氮的水平，促进刺槐的光合作用，为喷播基质促生菌提供了新的选择。
附图说明
图1是营养琼脂固体培养基上的库克氏菌属X-22、微杆菌属X-26和芽孢杆菌X-28菌落图；图中，A：库克氏菌属X-22；B：微杆菌属X-26；C：芽孢杆菌X-28。
具体实施方式
下面结合具体实施例进一步说明本发明，但这些实例并不用来限制本发明。下述实施例中所使用的方法如无特殊说明，均为常规方法。
实施例1
1)菌株的获得和鉴定
在岳阳市岳阳大道两侧坡地表面5cm根际土壤中采集土样，采用稀释涂布平板法，在35℃培养箱中，以营养琼脂固体培养基(NA培养基：蛋白胨10g； 牛肉粉3g；氯化钠5g；琼脂15g；无菌水1000mL)培养2～3d，肉眼观察挑取不同菌落，反复划线纯化得到若干不同种单菌落。
选单一菌落做成平板，送上海金域医学检验所测序，得16S rDNA基因序列，如SEQ ID NO.1所示。将所测16S rDNA基因序列与GenBank数据库中的序列进行BLAST比对。结果表明，该菌株与Kocuria polaris的相似度为99.32％。结合形态特征及16S rDNA基因序列分析，鉴定为库克氏菌属(Kocuria sp.)X-22。
选另一单一菌落做成平板，送上海金域医学检验所测序，得16S rDNA基因序列，如SEQ ID NO.2所示。将所测16S rDNA基因序列与GenBank数据库中的序列进行BLAST比对。结果表明，该菌株与的相似度为99.69％，与Microbacterium arabinogalactanolyticum的相似度为98.91％。结合形态特征及16S rDNA基因序列分析，鉴定为微杆菌属(Microbacterium sp.)X-26。
选再一单一菌落做成平板，送上海金域医学检验所测序，得16S rDNA基因序列，如SEQ ID NO.3所示。将所测16S rDNA基因序列与GenBank数据库中的序列进行BLAST比对。结果表明，该菌株与Bacillus megaterium的相似度为99.70％。结合形态特征及16S rDNA基因序列分析，鉴定为芽孢杆菌(Bacillus sp.)X-28。
2)库克氏菌属X-22、微杆菌属X-26和芽孢杆菌X-28的生理生化结果如表1所示，菌落图如图1所示。
表1菌株的生理生化结果
  X-22 X-26 X-28
葡萄糖发酵 ﹢无气泡 ﹢无气泡 ﹢无气泡
乳糖发酵 ﹢无气泡 ﹣无气泡 ﹢无气泡
淀粉水解 ﹣ ﹣ ﹢
吲哚试验 ﹣ ﹣ ﹢
甲基红试验 ﹢ ﹢ ﹢
V.P.试验 ﹣ ﹣ ﹣
柠檬酸盐试验 ﹢ ﹣ ﹣
硫化氢试验 ﹣ ﹣ ﹣
革兰氏染色 ﹢ ﹢ ﹢
菌落形态 球菌 杆菌 杆菌
实施例2
1、刺槐幼苗的培育
刺槐幼苗的培育在南京林业大学白马教学基地大棚温室内进行，空气湿度65％；CO 2浓度450ppm；最大光合有效辐射为1850μmol/(m 2·s)。确保各个盆栽每天接受光照强度和时间一致。为防止浇水过于频繁导致刺槐烂根，每次补充水分采用称重法浇水，保证每盆的土壤含水量均达到田间持水量的100％。
1)浸种：把筛好的刺槐种子用60℃的热水浸种(种子：水＝1:3)，待自然冷却继续浸种24h后，挑选其中已膨胀的种子进行催芽，剩余的种子用80℃的热水继续浸种，比例同上，待自然冷却24h后，挑选膨胀的种子进行催芽。通过逐次增温分批次浸种，既可以节约种子，又保证出苗整齐。浸种时，隔12h换一次冷水，去除水中杂质。
2)催芽：采用层积催芽法。将已经膨胀的种子混以3倍的湿沙(手松即散方可)。为了确保沙子的湿度，表面覆盖带有些许孔的保鲜膜，插入温度计，置于避光处。催芽3～4d，保持温度在20℃左右。
3)入盆与间苗：将处理过的基质混入盆中，栽入芽长1cm以上的幼苗，每盆栽入5株，每种处理设置3个平行；生长4周后进行间苗，每盆留3株长势一致的刺槐幼苗。
2、发酵菌液的制备：
1)取库克氏菌属X-22、微杆菌属X-26、芽孢杆菌X-28菌株，分别在营养琼脂固体培养基上，35℃活化24h；
2)将活化后的微杆菌属X-26、芽孢杆菌X-28菌株用接种环挑取一环菌泥分别加入到LB液体培养基中，库克氏菌属X-22接入到NA液体培养基中，35℃，200r/min恒温震荡24h制备种子液；
3)按3％的接种量取种子液接种于液体培养基(蛋白胨10g，酵母粉3g，氯化钠5g，无菌水1000mL，pH值5.6)，35℃，200r/min摇床振荡培养至OD 560为0.8-1.2(约36h)，得到发酵菌液；
4)使用前，用无菌水将步骤3)得到的发酵菌液稀释100倍后，等体积混合，使用。
3、盆栽实验
设置单菌对照组(X-22，X-26，和X-28的发酵菌液稀释液分别取60mL)，空白对照组(无菌液体发酵培养基)，混合菌组(三种发酵菌液稀释液各取20mL，共计60mL菌液)，通过根际土壤浇施的方法添加到栽植好的刺槐幼苗周围(前1-4周为间苗期，未施菌，后5-16周为施菌培养观察期)。各处理设置3个平行。
对盆栽进行一个季度的观测统计。于第8周开始将植株小心挖出，简单清理刺槐根部土壤并记录根瘤数量后复栽回花盆中；于第16周进行最后一次根瘤统计，记录根瘤个数和重量，结果如表2所示。
表2根瘤数、根瘤重和根干重结果
Figure PCTCN2020113434-appb-000001
从表2可知，根瘤最早于接三菌混合处理组形成，约在第8周和第12周之间，至最后一次采样已形成7个根瘤，数量较多。与空白组对照相比，施加三菌混合后刺槐的结瘤数量和结瘤重量有明显增加，其处理的刺槐根干重也有显著增加，相对空白组平均增长量为147.83％。可见，该混合菌能够促进刺槐结瘤固氮，促加速其根部生长发育，是很有前景的刺槐促生菌株配置模式。
4、促生实验
按照上述方法将活化的菌液混合后添加到栽植好的刺槐幼苗周围。从第一次间苗之后，每隔30d使用游标卡尺测量刺槐幼苗的地径；使用皮尺测量幼苗的苗高；于终止日，从每盆植株选取上中下位叶片共计10片采集，利用根系扫描仪测量叶面积，结果如表3所示。
表3促生结果
处理方式 地径(mm) 苗高(cm) 叶面积(cm 2)
混合菌 6.50±0.89 51.93±3.74 52.98±2.69
X-22 4.63±0.34 44.70±5.30 40.42±3.23
X-26 4.81±0.33 40.57±4.44 45.86±3.54
X-28 5.17±0.43 41.13±4.97 41.50±2.58
CK 4.75±0.38 40.04±5.76 43.53±2.21
地径用来表示树木的规格，长势好的植物一般地径都比较大。由表3可知，除X-22处理的刺槐幼苗的地径低于无菌幼苗，其余处理均高于无菌幼苗。其中混合菌处理的幼苗地径显著高于无菌幼苗，增加了36.84％(P＜0.05)。
苗高是植物形态学最基本的指标之一，可直观反映植被的生长状况，一般情况下，长势好的植物，其苗高都比较高。由表3可知，各配置土壤菌处理的苗高均高于无菌幼苗，苗高平均为44.58cm，平均增加量为11.34％。其中混合菌处理的苗高分别显著高于无菌幼苗增加了29.7％(P＜0.05)。
叶面积是与产量关系最密切的指标之一，植株的增产可由叶面积直观反映，一般情况下，适当大小的叶面积，既可以充分利用光照条件，又不影响光合作用。由表3可知，X-22、X-28处理的幼苗的叶面积低于无菌幼苗的叶面积。叶面积平均为45.19mm 2，平均增量为3.81％。其中混合菌处理的幼苗叶面积显著高于无菌幼苗的叶面积，增加21.71％(P＜0.05)。

Claims (9)

  1. 一种促进刺槐结瘤固氮的混合菌，其特征在于，由库克氏菌属(Kocuria sp.)X-22，微杆菌属(Microbacterium sp.)X-26，芽孢杆菌(Bacillus sp.)X-28组成；其中，
    所述库克氏菌属(Kocuria sp.)X-22，保藏于中国典型培养物保藏中心，保藏日期：2019年4月8日，保藏编号CCTCC No:M 2019237，保藏地址：中国武汉武汉大学；
    所述微杆菌属(Microbacterium sp.)X-26，保藏于中国典型培养物保藏中心，保藏日期：2019年4月8日，保藏编号CCTCC No:M 2019238，保藏地址：中国武汉武汉大学；
    所述芽孢杆菌(Bacillus sp.)X-28，保藏于中国典型培养物保藏中心，保藏日期：2019年4月8日，保藏编号CCTCC No:M 2019239，保藏地址：中国武汉武汉大学。
  2. 权利要求1所述的混合菌在促进刺槐结瘤固氮中的应用。
  3. 根据权利要求2所述的应用，其特征在于，将混合菌分别制备出发酵菌液，将各发酵液稀释并混合后直接在刺槐幼苗的根际土壤浇施。
  4. 根据权利要求3所述的应用，其特征在于，所述发酵菌液的制备方法为：
    1)取库克氏菌属X-22、微杆菌属X-26、芽孢杆菌X-28菌株，分别在营养琼脂固体培养基上，35℃活化24h；
    2)将活化后的微杆菌属X-26、芽孢杆菌X-28菌株用接种环挑取一环菌泥分别加入到LB液体培养基中，库克氏菌属X-22接入到NA液体培养基中，35℃，200r/min恒温震荡24h制备种子液；
    3)按3％的接种量取种子液接种于液体培养基，35℃，200r/min摇床振荡培养36h，得到发酵菌液；
    4)使用前，用无菌水将步骤3)得到的发酵菌液稀释后，等体积混合施用。
  5. 根据权利要求4所述的应用，其特征在于，步骤3)中，所述液体培养基为蛋白胨10g，酵母粉3g，氯化钠5g，无菌水1000mL，pH值5.6。
  6. 权利要求1所述的混合菌在促进刺槐生长中的应用。
  7. 根据权利要求6所述的应用，其特征在于，将混合菌分别制备出发酵菌液，将各发酵液稀释并混合后直接在刺槐幼苗的根际土壤浇施。
  8. 根据权利要求7所述的应用，其特征在于，所述发酵菌液的制备方法为：
    1)取库克氏菌属X-22、微杆菌属X-26、芽孢杆菌X-28菌株，分别在营养琼脂固体培养基上，35℃活化24h；
    2)将活化后的微杆菌属X-26、芽孢杆菌X-28菌株用接种环挑取一环菌泥分别加入到LB液体培养基中，库克氏菌属X-22接入到NA液体培养基中，35℃，200r/min恒温震荡24h制备种子液；
    3)按3％的接种量取种子液接种于液体培养基，35℃，200r/min摇床振荡培养36h，得到发酵菌液；
    4)使用前，用无菌水将步骤3)得到的发酵菌液稀释后，等体积混合施用。
  9. 根据权利要求8所述的应用，其特征在于，步骤3)中，所述液体培养基为蛋白胨10g，酵母粉3g，氯化钠5g，无菌水1000mL，pH值5.6。
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