大豆根瘤菌定向微生态研究与应用

doi:10.13304/j.nykjdb.2023.0758

摘要/Abstract

摘要：

我国大豆根瘤菌种质资源丰富，但其应用进展缓慢，接种面积仅占全国大豆总种植面积的1%~3%。造成该问题的原因主要是根瘤菌不能产芽孢，其抗逆能力较差，导致产品货架期短，应用效果不稳定。为实现根瘤菌产品多元化、功能化、差异化，提高产品活性与应用效果，使用定向微生态（directed micro-ecology， DME）体系优化培养策略，实现根瘤菌、芽孢杆菌的定向扩培及应用。通过单因素实验及响应面法获得根瘤菌最优培养基（25.5 mL·L^-1 糖蜜液、15.2 g·L^-1酵母膏、pH 6.98），在DME培菌机中使用该配方可以实现根瘤菌（100 L·min^-1，20 h）及芽孢杆菌（150 L·min^-1，14 h）菌量50倍以上的扩增，达到（20~50）×10⁸ CFU·mL^-1，且杂菌率控制在5%以下。盆栽接种实验表明，DME扩培菌株接种处理的效果显著优于实验室常规培养菌株的同等接种处理，这表明DME培菌机培养的功能微生物应用效果更好。根瘤菌与芽孢杆菌的复配组合可以协助根瘤菌更好地实现其共生功能，具体表现在苗期结瘤数增加、叶绿素含量增加，并且最终的产量与单接种菌株相比也增加了近20%。定向微生态（DME）体系通过定向营养和生长控制，使得目标功能微生物在含有多种微生物的微生态环境中，达到应用级别的产量和纯度，实现与纯培养菌株相同的功能，对于促进根瘤菌的产业化、加快功能菌株的推广应用具有重要意义和应用价值。

关键词: 定向微生态体系, 根瘤菌, 芽孢杆菌

Abstract:

China has abundant soybean-nodulating rhizobia germplasm resources， but its application progress is slow， which inoculation area is only 1%~3% of the total soybean planting area in the country. The main reason for the situation is that rhizobia cannot produce spores and have poor stress resistance， resulting in short product shelf life and unstable application effects. In order to diversify， functionalize and differentiate rhizobia products and improve product activity and application effects， a directed micro-ecology （DME） system was used to optimize the culture and successfully achieved the directing cultivation as well as application evaluation of rhizobia and bacillus strains. The optimal culture medium for rhizobia （25.5 mL·L^-1 molasses solution， 15.2 g·L^-1 yeast extract， pH 6.98） was obtained through single-factor experiment of culture medium and response surface method. The medium formulation was applied in DME incubator at proper ventilation volume and incubation time， which made 1 strain of rhizobia （100 L·min^-1， 20 h） and 3 strains of bacillus （150 L·min^-1， 14 h） to achieve more than 50-folds amplification of microbial quantities to reach （20~50）×10⁸CFU·mL^-1， and the rate of miscellaneous microorganism contamination was controlled below 5%. Pot experiments showed that the treatments of DME incubating strains were significantly better than the similar treatments of conventional cultured strains in the laboratory， which indicated that the DME incubator could better realize the application performances of functional microorganisms. The combination of rhizobia and bacillus could assist the rhizobia to better deliver its symbiotic function， which was specifically reflected in the increased number of nodules and chlorophyll content in the seedling stage， and the final yield was increased by nearly 20%， compared with single rhizobia strain. The DME system proposed in this study through the control of directed nutrition and growth strategies， allowed the target functional microorganisms to reach application-level yield and purity in a microecological environment containing a variety of microorganisms， achieving the same application performance as pure culture microbial product. It had important significance and application value to promote the industrialization of rhizobia， accelerating the promotion and application of functional strains.

Key words: directed micro-ecology system, rhizobia, bacillus

中图分类号:

S182

曹轶, 崔文靖, 马瑞强. 大豆根瘤菌定向微生态研究与应用[J]. 中国农业科技导报, 2024, 26(4): 215-224.

Yi CAO, Wenjing CUI, Ruiqiang MA. Research and Application of Directed Micro-Ecology in Soybean-Nodulating Rhizobia[J]. Journal of Agricultural Science and Technology, 2024, 26(4): 215-224.

图/表 8

表1 定向微生态培菌机DME-05参数

Table 1 Parameter of the directed micro-ecology microbial incubator DME-05

参数Parameter	数值Value
大小Size/mm	1 100×1 050×1 400
重量Weight/kg	300
功率Power/kW	6-8
电压Voltage/V	380/220
容量Volume/L	60
温度Temperature/℃	25~95
转速Rotation speed/（r·min^-1）	150~300
通气量Ventilation capacity/（L·min^-1）	50~150

表2 单因素水平设置

Table 2 Single factor level setting

水平Level

糖蜜液体积分数Volume fraction of molasses/%

酵母膏质量分数

Mass fraction of yeast/%

图1 培养基组分及初始pH对S. fredii GN85最终活菌数的影响注：不同小写字母表示不同处理间在P<0.05水平差异显著。

Fig. 1 Effects of medium composition and initial pH on viable cell concentration of S. fredii strain GN85Note： Different lowercase letters indicate significant differences between different treatments at P<0.05 level.

表3 单因素实验结果

Table 3 Result of Single factor test

编号 Code	变量水平Coded variable level			菌量 Cell concentration/（10⁸ CFU·mL^-1）
编号 Code	糖蜜液Molasses	酵母膏Yeast extract	pH	菌量 Cell concentration/（10⁸ CFU·mL^-1）
1	0	-1	1	31
2	-1	1	0	38
3	1	0	-1	35
4	0	1	1	33
5	0	0	0	48
6	1	0	1	37
7	-1	0	1	26
8	1	1	0	35
9	0	1	-1	34
10	1	-1	0	34
11	0	-1	-1	34
12	0	0	0	53
13	0	0	0	46
14	0	0	0	44
15	0	0	0	50
16	-1	-1	0	37
17	-1	0	-1	29

表4 响应面模型ANOVA检验

Table 4 ANOVA test for the response surface quadratic model

源Source	合计 Sum	自由度 df	均值 Mean	F值 F value	P值 P value
R² =0.932 2， R_Adj² =0.901 6
模型Model	622.2	9	69.13	4.145 0	0.037 1^*
A	190.1	1	190.10	11.400 0	0.011 8^*
B	28.13	1	28.13	1.686 0	0.235 2
C	12.5	1	12.50	0.749 5	0.415 3
AB	6.25	1	6.25	0.374 7	0.559 8
AC	1×10^-13	1	1.00×10^-13	0.000 0	1.000 0
BC	4	1	4.00	0.239 8	0.639 3
A²	11.12	1	11.12	0.666 6	0.441 1
B²	19.01	1	19.01	1.140 0	0.321 1
C²	331.6	1	331.60	19.888 0	0.002 9^*
残差Residual	116.8	7	16.68
失拟检验Lack of fit	56.75	3	18.92	1.261 0	0.399 9
纯差Pure error	60	4	15.00
总离差Cor total	738.9	16

图 2 糖蜜液、酵母膏用量及pH对GN85有效活菌数的响应面及其对应等高线

Fig. 2 Response surface plot and the corresponding contour plot of molasses， yeast and pH on viable cell concentration of GN85

图3 根瘤菌及芽孢杆菌在DME培菌机上不同通气量的培养情况A：根瘤菌的有效活菌数和污染率；B：3种芽孢杆菌的有效活菌率和污染率。菌株代号之后数字表示培养时间，不同小写字母表示不同处理间在P<0.05水平差异显著

Fig. 3 Culture status of rhizobia and bacillus strains under differnet ventilation volume by DME incubatorA： Final concentration and contamination rate of rhizobia； B： Final concentration and contamination rate of 3 bacillus strains.The culture time is fallowed by strain number. Different lowercase letters indicate significant differences between different treatments at P<0.05 level

图4 图接种DME扩培菌液及实验室纯培养菌液对苗期表型及最终产量的影响注：不同小写字母表示不同处理间在P<0.05水平差异显著。

Fig. 4 Effects of the inoculation with DME incubated medium and laboratory pure culture medium on seedling phenotype and final yieldNote： Different lowercase letters indicate significant differences between different treatments at P<0.05 level.

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