WO2021057441A1 - 一种根瘤促生菌微杆菌属x-18及其应用 - Google Patents

一种根瘤促生菌微杆菌属x-18及其应用 Download PDF

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WO2021057441A1
WO2021057441A1 PCT/CN2020/113411 CN2020113411W WO2021057441A1 WO 2021057441 A1 WO2021057441 A1 WO 2021057441A1 CN 2020113411 W CN2020113411 W CN 2020113411W WO 2021057441 A1 WO2021057441 A1 WO 2021057441A1
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microbacterium
robinia pseudoacacia
liquid
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fermentation broth
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庄家尧
刘超
王阳阳
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南京林业大学
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  • the invention belongs to the technical field of microorganisms, and specifically relates to a root nodule growth-promoting bacteria Microbacterium sp. X-18 and applications thereof.
  • 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 plants to ensure the nitrogen nutrition of plants; at the same time, the stomatal conductance of plants treated by microorganisms with a growth-promoting effect is increased Speed up 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 photosynthesis rate of plants and promotes plant photosynthesis; in addition, microorganisms can be used in their life activities. Decompose the incompatible minerals in the soil to degrade inorganic and organic pollutants. These all ensure the supply of nutrients required by plants in the microenvironment and the normal progress of plant life activities. Obviously, targeted screening of growth-promoting bacteria for specific plants is one of the keys to the wide application of the spray-seeding greening technology. At present, there are few reports on the selection of suitable nodule-promoting strains for different plants in China.
  • the purpose of the present invention is to provide a rhizobia growth-promoting bacterium Microbacterium X-18 to provide strain support for the establishment of a slope-protecting plant growth-promoting bacteria library.
  • Another object of the present invention is to provide the application of the above-mentioned Microbacterium X-18 in promoting nodulation and nitrogen fixation of Robinia pseudoacacia. It can provide high levels of nitrogen and promote nodulation and nitrogen fixation of Robinia pseudoacacia.
  • Another object of the present invention is to provide the application of the above-mentioned Microbacterium X-18 in promoting the growth of Robinia pseudoacacia. It can effectively promote the growth of ground diameter, seedling height and leaf area, and grows well.
  • a kind of nodule-promoting bacteria classified and named as Microbacterium sp. X-18, deposited in the China Type Culture Collection, preservation date: April 8, 2019, preservation number CCTCC No: M 2019236, Depositing address: Wuhan University, Wuhan, China.
  • the fermentation broth of Microbacterium X-18 is diluted and directly watered on the rhizosphere soil of Robinia pseudoacacia seedlings.
  • the application includes the following steps:
  • step 4 Before use, after diluting the fermentation broth obtained in step 3) with sterile water, add it to the planted locust seedling pot, 60 mL/pot.
  • the liquid medium is 10 g of peptone; 3 g of yeast powder; 5 g of sodium chloride; 1000 mL of sterile water; pH 5.6.
  • Microbacterium X-18 in promoting the growth of Robinia pseudoacacia.
  • the fermentation broth of Microbacterium X-18 is diluted and directly watered on the rhizosphere soil of Robinia pseudoacacia seedlings.
  • the application includes the following steps:
  • step 4) After diluting the fermentation bacteria liquid obtained in step 3) with sterile water, add it to the planted black locust seedling pot, 60 mL/pot.
  • the liquid culture medium is 10 g of peptone; 3 g of yeast powder; 5 g of sodium chloride; 1000 mL of sterile water; pH 5.6.
  • the microbacterium genus Rhizobium X-18 screened by the present invention can promote effective nitrogen fixation of Robinia pseudoacacia, provide nitrogen required for the growth of Robinia pseudoacacia, and make the growth-promoting root nodule bacteria and plants symbiotic nitrogen fix The ability has been obviously brought into play, and the development prospects are promising.
  • Figure 1 is a diagram of the colony of Microbacterium X-18 on a nutrient agar solid medium.
  • the soil samples were collected from the rhizosphere soil 5cm below the surface of the slope on both sides of Yueyang Avenue in Yueyang City, and the dilution spreading plate method was used in a 35°C incubator with nutrient agar solid medium (NA medium: peptone 10g; beef powder 3g ; Sodium chloride 5g; Agar 15g; Sterile water 1000mL) culture for 2 to 3 days, pick out different colonies by visual observation, and repeatedly streaking and purifying to obtain single colonies of different species.
  • NA medium peptone 10g
  • beef powder 3g agar
  • Sodium chloride 5g Agar 15g
  • Sterile water 1000mL Sterile water 1000mL
  • the main biological characteristics of this Microbacterium X-18 are: cultivated on nutrient agar medium (NA solid medium) for 24 hours at 35°C, as shown in Figure 1; colony characteristics: brown, moist and smooth, round, leathery Ran’s staining is blue-purple positive, and the shape is short rod; glucose fermentation test: -, no bubbles; lactose fermentation test:-no bubbles; starch hydrolysis test: -; indole test: -; methyl red test:- ; VP experiment: -; Citrate experiment: -; Hydrogen sulfide experiment: -.
  • Example 2 The ability of strains to promote nodulation and nitrogen fixation of Robinia pseudoacacia
  • Preparation of fermentation broth 1) Take Microbacterium X-18 from the slant, and activate it on a nutrient agar solid medium at 35°C for 24 hours; 2) Use an inoculating loop to pick up a loop of bacterial paste and add the activated strain Into 20mL LB liquid culture medium, 35°C, 200r/min constant temperature shaking for 24h to prepare seed liquid; 3) Inoculate the seed liquid into liquid medium at 3% of the inoculum amount, 35°C, 200r/min shaker culture to OD 560 is 0.8-1.2 to obtain a fermentation broth; 4) before use, the fermentation broth obtained in step 3) is diluted 100 times with sterile water, and sprayed directly in the potted black 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 2 that the ground diameter of the bacteria-added treatment group increased by 22.32% compared to the control group.
  • Seedling height is one of the most basic indicators of plant morphology, which can directly reflect the growth status of plants. In general, plants that grow well have relatively high seedling heights. It can be seen from Table 3 that the height of the seedlings of the treatment group increased by 27.87% compared to the control group.
  • 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 2 that the leaf area of the bacteria-added treatment group increased by 17.87% compared to the control group.

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Abstract

一种根瘤促生菌微杆菌属X-18及其应用。所述根瘤促生菌的分类命名为微杆菌属(Microbacterium sp.)X-18，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 2019236，保藏地址：中国武汉武汉大学。所述微杆菌属X-18应用于豆科植物刺槐中，能够促进刺槐有效固氮，提供刺槐生长所需的氮素，使根瘤促生菌与植物共生固氮能力得到明显发挥，从而促进刺槐植株的生长发育。

Description

一种根瘤促生菌微杆菌属X-18及其应用 技术领域
本发明属于微生物技术领域，具体涉及一种根瘤促生菌微杆菌属(Microbacterium sp.)X-18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物可直接吸收的离子态氮素，保证植物的氮素营养；同时，经具有促生作用的微生物处理过的植物气孔导度升高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。这些都保证了植物在微环境中所需各营养成分的供应以及植物各生命活动的正常进行。明显的，有针对性的筛选特定植物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，国内针对不同植物，筛选适宜的根瘤促生菌株鲜有报道。
发明内容
发明目的：针对现有技术存在的问题，本发明的目的是提供一种根瘤促生菌微杆菌属X-18，为护坡植物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微杆菌属X-18在促进刺槐结瘤固氮中的应用。能够提供高水平的氮素，促进刺槐结瘤固氮。本发明的再一目的是提供上述微杆菌属X-18在促进刺槐生长中的应用。能有效促进地径、苗高与叶面积的增长，长势良好。
技术方案：为了实现上述发明目的，本发明采用的技术方案如下：
一种根瘤促生菌，其分类命名为微杆菌属(Microbacterium sp.)X-18，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 2019236，保藏地址：中国武汉武汉大学。
微杆菌属X-18在促进刺槐结瘤固氮中的应用。
所述的应用，将微杆菌属X-18的发酵菌液稀释后直接在刺槐幼苗的根际土壤浇施。
所述的应用，包括以下步骤：
1)从斜面上取微杆菌属X-18，在营养琼脂固体培养基上，35℃活化24h；
2)将活化后的菌株用接种环挑取一环菌泥加入到LB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，添加到栽植好的刺槐幼苗盆栽中，60mL/盆。
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 5.6。
微杆菌属X-18在促进刺槐生长中的应用。
所述的应用，将微杆菌属X-18的发酵菌液稀释后直接在刺槐幼苗的根际土壤浇施。
所述的应用，包括以下步骤：
1)从斜面上取微杆菌属X-18，在营养琼脂固体培养基上，35℃活化24h；
2)将活化后的菌株用接种环挑取一环菌泥加入到LB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)用无菌水将步骤3)得到的发酵菌液稀释后，添加到栽植好的刺槐幼苗盆栽中，60mL/盆。
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 5.6。
有益效果：与现有技术相比，本发明筛选出的根瘤促生菌微杆菌属X-18，能够促进刺槐有效固氮，提供刺槐生长所需的氮素，使根瘤促生菌与植物共生固氮能力得到明显发挥，发展前景可期。
附图说明
图1是营养琼脂固体培养基上的微杆菌属X-18菌落图。
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。下述实施例中所用的材料、试剂等，如无特殊说明，均可从商业途径得到。
实施例1菌株的获得和鉴定
在岳阳市岳阳大道两侧坡地表面5cm下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，见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Microbacterium chocolatum的相似度为99.75％。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-18。
该微杆菌属X-18的主要生物学特征为：在营养琼脂培养基(NA固体培养基)35℃条件下培养24h，如图1所示；菌落特征：棕色，湿润光滑，圆形，革兰氏染色为蓝紫色阳性，形态为短杆状；葡萄糖发酵实验：﹣、无气泡；乳糖发酵实验：﹣无气泡；淀粉水解实验：﹣；吲哚试验实验：﹣；甲基红试验：﹣；V.P.实验：﹣；柠檬酸盐实验：﹣；硫化氢实验：﹣。
实施例2菌株促进刺槐结瘤固氮的能力
1、发酵液的制备：1)从斜面上取微杆菌属X-18，在营养琼脂固体培养基上，35℃活化24h；2)将活化后的菌株用接种环挑取一环菌泥加入到20mL LB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摇床振荡培养至OD 560为0.8-1.2，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，直接在栽植好的刺槐幼苗盆栽中喷施。
2、盆栽实验：按60mL/盆，取稀释后的发酵菌液添加到栽植好的刺槐幼苗周围(实际种植期是4个月，第一个月为间苗期，未施菌，间苗期后施菌开始计算)。并设置无菌水为空白对照组(CK)，经过一个季度观测分析刺槐的结瘤固氮能力。
对盆栽进行观测统计，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1所示。
表1 刺槐的根瘤数、根瘤重、根干重结果
Figure PCTCN2020113411-appb-000001
从表1可知，与空白组对照相比，加入微杆菌属X-18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121.74％。可见该菌株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菌株。
3、促生实验：按60mL/盆，取稀释后的发酵菌液添加到栽植好的刺槐幼苗周围。一个月之后对刺槐幼苗进行间苗，保证每盆幼苗数量的整齐度。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2所示。
表2 刺槐幼苗一个季度生长指标
Figure PCTCN2020113411-appb-000002
注：P＜0.05。
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2可知，加菌处理组的地径相对对照组增加了22.32％。
苗高是植物形态学最基本的指标之一，可直观反映植物的生长状况，一般情况下，长势好的植物，其苗高都比较高。由表3可知，加菌处理组的苗高相对对照组增加了27.87％。
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2可知，加菌处理组的叶面积相对对照组增加了17.87％。

Claims (9)

  1. 一种根瘤促生菌，其分类命名为微杆菌属(Microbacterium sp.)X-18，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 2019236，保藏地址：中国武汉武汉大学。
  2. 权利要求1所述的微杆菌属X-18在促进刺槐结瘤固氮中的应用。
  3. 权利要求2所述的应用，其特征在于，将微杆菌属X-18的发酵菌液稀释后直接在刺槐幼苗的根际土壤浇施。
  4. 权利要求3所述的应用，其特征在于，包括以下步骤：
    1)从斜面上取微杆菌属X-18，在营养琼脂固体培养基上，35℃活化24h；
    2)将活化后的菌株用接种环挑取一环菌泥加入到LB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，添加到栽植好的刺槐幼苗盆栽中，60mL/盆。
  5. 根据权利要求4所述的应用，其特征在于，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 5.6。
  6. 权利要求1所述的微杆菌属X-18在促进刺槐生长中的应用。
  7. 权利要求6所述的应用，其特征在于，将微杆菌属X-18的发酵菌液稀释后直接在刺槐幼苗的根际土壤浇施。
  8. 权利要求7所述的应用，其特征在于，包括以下步骤：
    1)从斜面上取微杆菌属X-18，在营养琼脂固体培养基上，35℃活化24h；
    2)将活化后的菌株用接种环挑取一环菌泥加入到LB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)用无菌水将步骤3)得到的发酵菌液稀释后，添加到栽植好的刺槐幼苗盆栽中，60mL/盆。
  9. 根据权利要求7所述的应用，其特征在于，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 5.6。
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