砂质潮土高效溶磷菌的筛选鉴定、条件优化及应用

doi:10.13560/j.cnki.biotech.bull.1985.2020-0550

摘要/Abstract

摘要：

从砂质潮土中分离筛选出一株高效溶磷菌,并初探其对土壤磷素形态变化及小麦生长的影响,旨在为其在实际生产中的应用奠定理论基础。利用PKO培养基从砂质潮土中分离几株溶磷菌,对比其溶磷能力,筛选出一株高效溶磷菌,综合菌株形态、生理生化特性以及16S rDNA 分子学鉴定菌株种属,对其溶磷条件进行优化,并通过盆栽实验探索菌株在土壤中的溶磷过程,验证菌株促生效果。结果显示,筛选出一株高效溶磷菌W6,经鉴定为弯曲芽孢杆菌（Bacillus flexus）;摇瓶实验结果表明：当培养时间为24 h、初始pH 6.0、装液量75 mL/250 mL、碳源为木糖、氮源为硝酸钾时,该菌株溶磷量达到最大;小麦盆栽实验结果表明：与对照相比,接种菌株W6的土壤H₂O-P、NaHCO₃-Po、NaHCO₃-Pi、NaOH-Po、NaOH-Pi含量分别增加了127.3%、80.0%、54.5%、11.8%和20.0%,而难溶性HCl-P、浓HCl-Po、浓HCl-Pi、浓H₂SO₄-P含量分别降低了10.9%、23.0%、42.1%和33.3%,接种菌株W6的小麦根长、根表面积、根体积、根尖数、根直径分别显著增加了66.8%、57.0%、50.0%、13.6%和9.1%;小麦干重、氮、磷及钾含量分别显著增加了70.5%、32.1%、24.7%、94.2%和34.4%。菌株W6能够促进磷素形态由难溶性向有效性进行转化,对小麦根系的生长、发育具有明显的促进作用,提高植物对磷素的吸收及利用效率,在农业生产上有着较大的应用潜力。

关键词: 小麦, 溶磷菌, 砂质潮土, 条件优化, 磷素分级

Abstract:

The objectives of this work are to isolate an efficient phosphate solubilizing bacterium from the sandy fluvo aquic soil,to study its effect on the change of soil phosphorus form and the growth of wheat,and to provide the theoretical basis for its application in practical production. PKO medium was used to isolate several phosphate solubilizing bacteria from sandy fluvo aquic soil,and their phosphate solubilizing abilities were compared to screen out an efficient phosphate solubilizing bacterium. The species of the bacterium was identified by combining the morphological,physiological and biochemical characteristics of the bacteria as well as 16S rDNA molecular analysis. The phosphate solubilizing conditions were optimized,and phosphorus-dissolving process was explored and growth-promoting effect of the bacterium in the soil was verified by pot experiment. As results,an efficient phosphate solubilizing bacterium W6 was screened and identified as Bacillus flexus. The results of shake flask experiment showed that the phosphorus dissolving capacity of the strain reached the maximum when the culture time was 24 hours,the initial pH was 6.0,the liquid content was 75 mL/250 mL,the carbon source was xylose,and the nitrogen source was potassium nitrate. The results from wheat pot experiment demonstrated that the contents of H₂O-P,NaHCO₃-Po,NaHCO₃-Pi,NaOH-Po,and NaOH-Pi increased by 127.3%,80.0%,54.5%,11.8%,20.0%,respectively,while the contents of hard-soluble HCl-P,concentrated HCl-Po,concentrated HCl-Pi,and concentrated H₂SO₄-P decreased by 10.9%,23.0%,42.1%,33.3%,respectively,when compared with the control. The root length,root surface area,root volume,root tip number and root diameter of wheat inoculated with the strain W6 increased 66.8%,57.0%,50.0%,13.6% and 9.1%,respectively,and the dry weight,nitrogen,phosphorus and potassium contents of wheat increased 70.5%,32.1%,24.7%,94.2% and 34.4%,respectively. In conclusion,the strain W6 may promote the transformation of phosphorus form from insoluble to effective,also promote the growth and development of wheat root system,improve the absorption and utilization efficiency of phosphorus by plants,and thus presents great application potential in agricultural production.

Key words: wheat, phosphate solubilizing bacteria, sandy fluvo aquic soil, condition optimization, phosphorus fractionation

魏畅, 戚秀秀, 吴越, 刘晓丹, 王祎, 姜瑛, 柳海涛. 砂质潮土高效溶磷菌的筛选鉴定、条件优化及应用[J]. 生物技术通报, 2021, 37(4): 85-95.

WEI Chang, QI Xiu-xiu, WU Yue, LIU Xiao-dan, WANG Yi, JIANG Ying, LIU Hai-tao. Screening,Identification,Condition Optimization and Application of Efficient Phosphate Solubilizing Bacteria in Sandy Fluvo Aquic Soil[J]. Biotechnology Bulletin, 2021, 37(4): 85-95.

图/表 12

图1 菌株溶磷效果图中误差线表示标准偏差。小写字母表示不同溶磷菌间差异达到（P < 0.05）显著水平,下同

Fig.1 Phosphate solubilizing efficiency of strains The error line in the figure refers to the standard deviation. The lowercase letters indicate that the difference among different phosphate-solubilizing bacteria reached a significant level (P < 0.05). The same below

图2 菌株 W6 的形态观察及革兰氏染色

Fig.2 Morphological observation and Gram staining of strain W6

表1 W6菌株的生理生化特性

Table 1 Physiological characteristics of strain W6

项目Item	结果Results	项目Projects	结果Results
革兰氏染色 Gram staining	+	淀粉水解 Starch hydrolysis	+
好氧性实验 Aerobic test	兼性厌氧 Facultative anaerobic	柠檬酸盐利用 Citrate utilization	-
接触酶实验 Catalase test	+	硝酸盐还原 Nitrate reduction	+
甲基红（M.R）反应Methyl red reaction	-	V-P实验 V-P test	+

图3 基于菌株W6和相关菌株的16S rDNA 序列采用邻接法建立的系统发育树标尺代表每1 000个核苷中有2个核苷替代

Fig. 3 Phylogenetic tree that based on 16S rDNA sequences of strain W6 and related strains Each scale refers to that there are two nucleoside substitutions per 1 000 nucleosides

图4 不同培养时间对W6菌株溶磷量的影响

Fig.4 Effect of different incubation time on the phosp-hate-solublizing efficiency of strain W6

图5 不同碳源对W6菌株溶磷量的影响

Fig.5 Effect of different carbon sources on the phosphate-solublizing efficiency of strain W6

图6 不同氮源对W6菌株溶磷量的影响

Fig.6 Effect of different nitrogen sources on the phosp-hate-solublizing efficiency of strain W6

图7 不同pH对W6菌株溶磷量的影响

Fig.7 Effect of different pH on the phosphate-solublizing efficiency of strain W6

图8 不同装液量对W6菌株溶磷量的影响

Fig. 8 Effect of different liquid volume on the phospho-rus efficiency of strain W6

表2 接种菌株W6对土壤磷素形态的影响

Table 2 Effects of innoculated strain W6 on the forms of phosphorus

处理Treatments		不同提取态磷素含量Contents of phosphorus in different extraction states/（g·kg^-1）								回收率 Recovery rate
处理Treatments	H₂O-P	NaHCO₃-Po	NaHCO₃-Pi	NaOH-Po	NaOH-Pi	HCl-P	浓HCl-Po Concentrated HCl-Po	浓HCl-Pi Concentrated HCl-Pi	浓H₂SO₄-P Concentrated H₂SO₄-P	回收率 Recovery rate
CK	0.11±0.02	0.35±0.10	0.22±0.00	0.17±0.02	0.15±0.03	1.56±0.07	0.74±0.07^*	0.57±0.04^*	0.09±0.02	98%
W6	0.25±0.00**	0.63±0.10^**	0.34±0.02	0.19±0.01	0.18±0.02	1.39±0.16	0.57±0.12	0.33±0.09	0.06±0.01	96%

表3 接种菌株W6对小麦根系的影响

Table 3 Effects of strain W6 on the root system of wheat

处理Treatment	根长Root length/cm	根表面积Root surface area/cm²	根体积Root volume/cm³	根尖数Number of root shoots/个	根直径Root diameter/cm
CK	584.58±58.81	43.73±1.40	0.26±0.04	2578±71.91	0.22±0.15
W6	975.06±22.21**	68.66±1.75**	0.39±0.02**	2929±32.19*	0.24±0.02*

表4 接种菌株W6对小麦植株的影响

Table 4 Effects of strain W6 on wheat

处理 Treatment	干重 Dry weight/g	株高 Plant height/cm	全氮 Total nitrogen/（g·kg^-1）	全磷 Total phosphorus/（g·kg^-1）	全钾 Total potassium/（g·kg^-1）
CK	1.46±0.07	16.52±1.68	1.86±0.17	1.71±0.56	6.68±0.58
W6	2.49±0.77**	21.83±0.31*	2.32±0.06*	3.32±0.55**	8.98±0.19**

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