产IAA兼具溶磷解钾高效促生菌的筛选、鉴定及其广谱性应用

doi:10.13560/j.cnki.biotech.bull.1985.2021-1557

摘要/Abstract

摘要：

从植株根际筛选出具有多种功能的促生菌种,对其促生功能进行探究,为实现农业“增产提效”提供优质菌肥种质资源。从根际土壤中分离出菌株,用Salkowski比色法测定产吲哚乙酸（IAA）能力,并用钼锑抗比色法和火焰光度法测定溶磷解钾能力,筛选出高效促生菌株并进行形态、生理生化及16S rDNA系统鉴定,探究其产IAA最适条件,进而通过盆栽和大田试验研究其实际应用效果。实验筛选出一株产IAA达61.71 mg/L、解钾能力达17.57 mg/L、解有机、无机磷能力分别达到98.25 mg/L和 0.64 mg/L的威兹曼芽胞杆菌（Bacillus wiedmannii）YC9;其产IAA能力在装液量为150/250 mL、pH为7-9、碳源为葡萄糖、氮源为蛋白胨时最优;盆栽试验中通过主成分分析（PCA）结合相关性分析发现YC9可以通过提高土壤中IAA以及速效磷钾含量,从而促进烟草根系及地上部的生长。在烟草和小麦的大田试验中,烤烟的产量和上等烟的比例分别增加了14.3%和9.6%,小麦增产了43.8%,表明YC9在烟草与小麦种植中均有良好的应用效果。YC9是一株兼具产IAA、解磷、解钾的多功能促生菌,能够在河南省潮土种植区提高经济作物的产量和品质,增加粮食作物的产量,具有广谱性应用前景。

关键词: 潮土, 根际促生菌, 烟草, 小麦, 威兹曼芽胞杆菌

Abstract:

We screened the growth-promoting bacteria species from plant rhizosphere and found out their growth-promoting ability under laboratory and field conditions,which may provide a potential bacterial fertilizer for promoting the production and efficiency in agriculture generally. The strains were isolated from rhizosphere soil,the IAA production of these strains was determined by Salkowski colorimetry,and the phosphorus- and potassium-dissolving abilities were determined by molybdenum antimony resistance colorimetry and flame photometry. The high-efficiency growth-promoting strains were identified in this study based on the morphology,physiology and biochemistry and 16S rDNA. The optimal conditions of these strains to produce IAA were explored and further applied to the pot and field experiments for studying its actual application effects. A strain of Bacillus wiedmannii YC9 was selected,it produced 61.71 mg/L of IAA,dissolved 17.57 mg/L of potassium and 98.25 mg/L and 0.64 mg/L of inorganic phosphorus and organic phosphorus. The IAA production of YC9 reached the peak under the conditions that the liquid volume was 150/250 mL,the pH was 7-9,the carbon source was glucose,and the nitrogen source was peptone. In the pot experiment,principal component analysis（PCA）combined with correlation analysis indicated that YC9 promoted the growth of tobacco roots and shoots by increasing IAA and available phosphorus and potassium in the soil. In the field trials of tobacco and wheat,the yield of flue-cured tobacco and the proportion of high-quality tobacco increased by 14.3% and 9.6%,respectively,and the yield of wheat increased by 43.8%,indicating that YC9 had a good application effect in both tobacco and wheat cultivation. YC9 has been identified as a multifunctional growth-promoting bacterium that can produce IAA,hydrolyze phosphorus and potassium. It improves the yield and quality of crops efficiently in the fluvo aquic soil. Our study shows it an outstanding microbial resource of growth-promoting bacterial fertilizer with a broad-spectrum application prospect.

Key words: fluvo aquic soil, rhizosphere growth-promoting bacteria, tobacco, wheat, Bacillus wiedmannii

张昊鑫, 王中华, 牛兵, 郭慷, 刘璐, 姜瑛, 张仕祥. 产IAA兼具溶磷解钾高效促生菌的筛选、鉴定及其广谱性应用[J]. 生物技术通报, 2022, 38(5): 100-111.

ZHANG Hao-xin, WANG Zhong-hua, NIU bing, GUO Kang, LIU Lu, JIANG Ying, ZHANG Shi-xiang. Screening,Identification and Broad-spectrum Application of Efficient IAA-producing Bacteria Dissolving Phosphorus and Potassium[J]. Biotechnology Bulletin, 2022, 38(5): 100-111.

图/表 15

表1 供试土壤基本理化性状

Table 1 Basic physical and chemical properties of tested soil

有机质Organic matter/%	全磷Total P/（g·kg^-1）	有效磷Available P/（mg·kg^-1）	全钾Total K/（g·kg^-1）	有效钾Available K/（mg·kg^-1）	pH
1.11	0.80	15.93	18.56	125.33	7.8

表2 菌株生长及产IAA能力条件优化

Table 2 Optimization of conditions for strain growth and IAA production capacity

不同条件Different condition	装液量 Liquid loading/（mL·250 mL^-1）	pH	氮源Nitrogen source	碳源Carbon source
1	25	4	硝酸钾	葡萄糖
2	50	5	硫酸铵	木糖
3	75	6	硝酸铵	蔗糖
4	100	7	酵母粉	果糖
5	150	8	谷氨酸	甘露醇
6		9	尿素	乳糖
7		10	蛋白胨	麦芽糖

表3 麦田供试土壤基本性质

Table 3 Basic properties of test soil in wheat field

有机质 Organic matter/%	全磷 Total P/（g·kg^-1）	有效磷 Available P/（mg·kg^-1）	全钾 Total K/（g·kg^-1）	有效钾 Available K/（mg·kg^-1）	全氮 Total N/（g·kg^-1）
1.91	0.29	2.44	19.56	30.42	0.46

表4 YC9各项促生能力

Table 4 Various growth promoting abilities of YC9

菌株 Strain	产IAA能力 IAA content/（mg·L^-1）	解钾能力 Available K content/（mg·L^-1）	解无机磷能力 Available P content/（mg·L^-1）	解有机磷能力 Available P content/（mg·L^-1）
YC9	61.71	17.57	98.25	0.64

图1 菌株YC9的菌落图（A）,革兰氏染色图（B）及电镜图（C）

Fig. 1 Colony diagram（A）,Gram staining diagram（B）and electron microscope diagram（C）of strain YC9

图2 YC9 菌株 16S rDNA 基因序列的系统发育树标尺代表每10 000个核苷中有5个核苷替代

Fig. 2 Phylogenetic tree of 16S rDNA gene sequence of YC9 strain The scale indicates 5 nucleoside substitutions per 10 000 nucleosides

表5 YC9菌株的生理生化特性

Table 5 Physiological and biochemical characteristics of YC9 strain

项目 Item	结果 Result	参考菌株 Reference strain	项目 Item	结果 Result	参考菌株 Reference strain
革兰氏染色 Gram stain	+	+	淀粉水解 Amylohydrolysis test	+	+
好氧性试验 Aerobic test	兼性厌氧	兼性厌氧	明胶液化 Gelatin liquefaction test	+	+
接触酶试验 Catalase	+	+	硝酸盐还原 Nitrate reduction test	+	+
甲基红（M.R）反应 Methyl red test	-	-	柠檬酸盐利用 Citrate utilization	-	+（55）
V-P试验 Voges-Proskauer test	+	+

图3 不同培养条件对YC9菌株产IAA能力和生长状况（OD600）的影响不同字母表示不同条件对YC9产IAA能力和生长状况的影响

Fig. 3 Effects of different culture conditions on the IAA production capacity and growth status（OD600）of YC9 strain Different letters indicate the effects of different conditions on the IAA production capacity and growth status of YC9

表6 接种菌株YC9 30 d后对土壤IAA以及速效磷钾含量的影响

Table 6 Effects of inoculating strain YC9 on the soil IAA and available phosphorus and potassium contents after 30 d

处理Treatment	IAA/（mg·kg^-1）	速效钾Available K/（mg·kg^-1）	速效磷Available P/（mg·kg^-1）
CK	0.26±0.04	135.00±7.35	5.04±0.08
YC9	0.94±0.05^**	149.00±1.73^*	6.11±0.32^*

图4 YC9对烟草盆栽根系（A）和植株（B）的影响

Fig. 4 Effect of YC9 on the root（A）and plant（B）of tobacco in the pot experiment

图5 YC9处理下烟草盆栽土壤及烟草各指标变化的主成分分析（A、B、C）和随机森林图（D）

Fig. 5 Principal component analysis（A,B,and C）and random forest map（D）of tobacco potted soil and tobacco indexes under YC9 treatment

表7 YC9处理下烟草盆栽土壤及烟草各指标变化相关性分析

Table 7 Correlation analysis of changes of tobacco pot soil and tobacco indexes under YC9 treatment

标标Index	根总长 Root length/cm	根表面积 Root surface area/cm²	根体积 Root volume/cm³	根尖数 Number of root tips	根平均直径 Average root diameter/mm	根分枝数 Number of root branches	植株鲜重 Plant fresh weight/g	株高 Plant height/cm	SPAD	植株全氮 Plant total N/（g·kg^-1）	植株全磷 Plant total P/（g·kg^-1）	植株全钾 Plant total K/（g·kg^-1）
土壤速效磷 Soil P/（mg·kg^-1）	0.855*	0.802	0.880*	0.717	0.479	0.613	0.816*	0.793	0.824*	0.755	0.783	0.771
土壤速效钾 Soil K/（mg·kg^-1）	0.903*	0.867*	0.881*	0.768	0.638	0.719	0.994**	0.988**	0.934**	0.538	0.959**	0.877*
土壤IAA/（mg·kg^-1）	0.855	0.802	0.880	0.717	0.479	0.613	0.816	0.793	0.824	0.755	0.783	0.771

图6 YC9菌剂对烟田土壤理化性质（A）以及烟草品质的影响（B）

Fig. 6 Effects of YC9 bacterial agent on the physical and chemical properties of tobacco field soil（A）and tobacco yield and quality（B）

表8 YC9处理对烤烟产量性状的影响

Table 8 Effects of YC9 treatment on the yield characters of flue-cured tobacco

处理 Treatments	单叶重Single leaf weight/g			产量 Yield/g	上等烟比例 Proportion of superior tabacco/%
处理 Treatments	下桔二（X2F）	中桔三（C3F）	上桔二（B2F）	产量 Yield/g	上等烟比例 Proportion of superior tabacco/%
CK	7.2	10.7	12.5	2015.0	58.2
YC9	7.5	11.9	13.4	2302.4	63.8

图7 YC9菌剂对麦田土壤理化性质（A）以及小麦产量的影响（B）

Fig. 7 Effect of YC9 bacterial agent on the physical and chemical properties of wheat field soil（A）and wheat yield（B）

参考文献 37

[1]	陈海生, 魏跃伟, 刘国顺, 等. 河南省烤烟种植区土壤综合肥力评价[J]. 水土保持研究, 2011, 18(3):131-136, 0, 2.
	Chen HS, Wei YW, Liu GS, et al. Studies on comprehensive evaluation of soil fertility in Henan tobacco planting regions based on fuzzy mathematics and principal component analysis[J]. Res Soil Water Conserv, 2011, 18(3):131-136, 0, 2.
[2]	马常宝, 卢昌艾, 任意, 等. 土壤地力和长期施肥对潮土区小麦和玉米产量演变趋势的影响[J]. 植物营养与肥料学报, 2012, 18(4):796-802.
	Ma CB, Lu CG, Ren Y, et al. Effect of soil fertility and long-term fertilizer application on the yields of wheat and maize in fluvo-aquic soil[J]. Plant Nutr Fertil Sci, 2012, 18(4):796-802.
[3]	陈越, 李虎林, 朱诗苗, 等. 产吲哚乙酸(IAA)促生菌的分离鉴定及对烟草种子萌发和幼苗生长发育的影响[J]. 作物杂志, 2020(2):176-181.
	Chen Y, Li HL, Zhu SM, et al. Isolation and identification of IAA-producing rhizobacteria and its effects on seed germination and seedling growth of tobacco[J]. Crops, 2020(2):176-181.
[4]	李培培, 汪强, 等. 不同还田方式对砂质潮土理化性质及微生物的影响[J]. 生态学报, 2017, 37(11):3665-3672.
	Li PP, Wang Q, et al. Effects of the return of organic materials on soil physical and chemical properties and bacterial number in sandy soil[J]. Acta Ecol Sin, 2017, 37(11):3665-3672.
[5]	慕兰, 慕琦. 复合微生物肥料对潮土理化性质和小麦产量的影响[J]. 农业科技通讯, 2018(11):30-32.
	Mu L, Mu Q. Effect of compound microbial fertilizer on physical and chemical properties of alluvial soil and wheat yield[J]. Bullenin Agric Sci Technol, 2018(11):30-32.
[6]	王豹祥, 李富欣, 张朝辉, 等. 应用PGPR菌肥减少烤烟生产化肥的施用量[J]. 土壤学报, 2011, 48(4):813-822.
	Wang BX, Li FX, Zhang CH, et al. Effect of application of PGPR on chemical fertilizer application rate for flue-cued tobacco[J]. Acta Pedol Sin, 2011, 48(4):813-822.
[7]	王政, 敖金成, 张真, 等. 微生物菌肥对烤烟生长发育和品质的影响[J]. 湖北农业科学, 2018, 57(10):79-82, 87.
	Wang Z, Ao JC, Zhang Z, et al. Effects of different microbial fertilizer on growth and development and quality of flue-cured tobacco[J]. Hubei Agric Sci, 2018, 57(10):79-82, 87.
[8]	聂登壁, 施文. 根际促生菌在烟草生产中的应用研究进展[J]. 现代农业科技, 2018(10): 8, 10.
	Nie DB, Shi W. Advances on application of PGPR in tobacco production[J]. Mod Agric Sci Technol, 2018(10): 8, 10.
[9]	Lucy M, Reed E, Glick BR. Applications of free living plant growth-promoting rhizobacteria[J]. Antonie Van Leeuwenhoek, 2004, 86(1):1-25. pmid: 15103234
[10]	张典利, 孟臻, 亓文哲, 等. 植物根际促生菌的研究与应用现状[J]. 世界农药, 2018, 40(6):37-43, 50.
	Zhang DL, Meng Z, Qi WZ, et al. The research and application status of plant growth promoting rhizobacteria[J]. World Pestic, 2018, 40(6):37-43, 50.
[11]	中国科学院南京土壤研究所微生物室. 土壤微生物研究法[M]. 北京: 科学出版社, 1985.
	Laboratory of Microbiology, Nanjing Institute of soil, Chinese Academy of Sciences. Soil microbial research method[M]. Beijing: Science Press, 1985.
[12]	赵小蓉, 林启美, 孙焱鑫, 等. 细菌解磷能力测定方法的研究[J]. 微生物学通报, 2001, 28(1):1-4.
	Zhao XR, Lin QM, Sun YX, et al. The methods for quantifying capacity of bacteria in dissolving p compounds[J]. Microbiology, 2001, 28(1):1-4.
[13]	赵斌, 何绍江. 微生物学实验[M]. 北京: 科学出版社, 2002.
	Zhao B, He SJ. Microbiology experiment[M]. Beijing: Science Press, 2002.
[14]	Libbert E, Manteuffel R. Interactions between plants and epiphytic bacteria regarding their auxin metaholism[J]. Physiol Plant, 1970, 23(1):93-98. doi: 10.1111/j.1399-3054.1970.tb06395.x URL
[15]	Glickmann E, Dessaux Y. A critical examination of the specificity of the salkowski reagent for indolic compounds produced by phytopathogenic bacteria[J]. Appl Environ Microbiol, 1995, 61(2):793-796. doi: 10.1128/aem.61.2.793-796.1995 URL
[16]	东秀珠, 蔡妙英. 常见细菌系统鉴定手册[M]. 北京: 科学出版社, 2001.
	Dong XZ, Cai MY. Manual for identification of common bacterial systems[M]. Beijing: Science Press, 2001.
[17]	郭素枝. 扫描电镜技术及其应用[M]. 厦门: 厦门大学出版社, 2006.
	Guo SZ. Scanning electron microscopy and its application[M]. Xiamen, China: Xiamen University Press, 2006.
[18]	夏北成. 分子生物学方法在微生物生态学中的应用[J]. 中山大学学报:自然科学版, 1998, 37(2):97-101.
	Xia BC. Application of molecular methods in microbial ecology[J]. Acta Sci Nat Univ Sunyatseni, 1998, 37(2):97-101.
[19]	Monis PT, Giglio S, Saint CP. Comparison of SYTO9 and SYBR Green I for real-time polymerase chain reaction and investigation of the effect of dye concentration on amplification and DNA melting curve analysis[J]. Anal Biochem, 2005, 340(1):24-34. doi: 10.1016/j.ab.2005.01.046 pmid: 15802126
[20]	毛小芳, 胡锋, 陈小云, 等. 不同土壤水分条件下华美新小杆线虫对枯草芽孢杆菌数量、活性及土壤氮素矿化的影响[J]. 应用生态学报, 2007, 18(2):405-410.
	Mao XF, Hu F, Chen XY, et al. Effects of Caenorhabditis elegans on Bacillus subtilis number and activity and soil nitrogen mineralization under different soil moisture conditions[J]. Chin J Appl Ecol, 2007, 18(2):405-410.
[21]	李金昶, 石晶, 等. 高效液相色谱法分离和测定3种植物内源激素[J]. 分析化学, 1994, 22(8):801-804.
	Li JC, Shi J, et al. Separation and determination of three kinds of plant hormones by high-performance liquid chromatography[J]. Chin J Anal Chem, 1994, 22(8):801-804.
[22]	鲍士旦. 土壤农化分析[M]. 3版. 北京: 中国农业出版社, 2000.
	Bao SD. Soil and agricultural chemistry analysis[M]. 3rd ed.ed. Beijing: Chinese Agriculture Press, 2000.
[23]	陈义强, 刘国顺, 习红昂. 基于烟叶品质指数的氮、磷、钾施肥模型[J]. 植物营养与肥料学报, 2013, 19(3):632-643.
	Chen YQ, Liu GS, Xi HA. Fertilization model of nitrogen, phosphorus and potassium based on quality index of tobacco leaves[J]. J Plant Nutr Fertil, 2013, 19(3):632-643.
[24]	张喜峰, 张立新, 高梅, 等. 不同氮肥形态和腐殖酸对陕西典型生态区烤烟化学成分和产质量的影响[J]. 草业学报, 2013, 22(6):60-67.
	Zhang XF, Zhang LX, Gao M, et al. Effects of different nitrogen fertilizer types and humic acid(HA)on chemical composition, yield and quality of flue-cured tobacco traits in typical ecological zones of Shaanxi Province[J]. Acta Prataculturae Sin, 2013, 22(6):60-67.
[25]	熊伟东, 刘晔, 王国文, 等. 促生菌剂在砂质潮土麦田的应用效果[J]. 麦类作物学报, 2016, 36(7):945-950.
	Xiong WD, Liu Y, Wang GW, et al. Effects of plant growth-promoting rhizobacteria agents application on wheat in sandy soil[J]. J Triticeae Crops, 2016, 36(7):945-950.
[26]	刘长征, 姜晓琳, 蔡启忠, 等. 何首乌根际促生菌的筛选及其对何首乌种子萌发的影响[J]. 中国中药杂志, 2021, 46(20):5247-5252.
	Liu CZ, Jiang XL, Cai QZ, et al. Screening of plant growth-promoting rhizobacteria and its effect on seed germination of Polygonum multiflorum[J]. China J Chin Mater Med, 2021, 46(20):5247-5252.
[27]	吴婧, 聂彩娥, 朱媛媛, 等. 一株兼具产IAA能力纤维素降解菌的筛选、鉴定及条件优化[J]. 生物技术通报, 2020, 36(12):54-63. doi: 10.13560/j.cnki.biotech.bull.1985.2020-0454
	Wu J, Nie CE, Zhu YY, et al. Isolation, identification of a cellulose-degrading bacterium with IAA-producing ability and optimization of its culture conditions[J]. Biotechnol Bull, 2020, 36(12):54-63.
[28]	刘晓. 微生物菌肥在农业生产中的应用研究[J]. 河南农业, 2021(17):14-15.
	Liu X. Application of microbial fertilizer in agricultural production[J]. Agric Henan, 2021(17):14-15.
[29]	Pandey A, Tamta S, Giri D. Role of auxin on adventitious root formation and subsequent growth of cutting raised plantlets of Ginkgo biloba L[J]. Int J Biodivers Conserv, 2011, 3:142-146.
[30]	Miller RA, Beno SM, Kent DJ, et al. Bacillus wiedmannii sp. nov., a psychrotolerant and cytotoxic Bacillus cereus group species isolated from dairy foods and dairy environments[J]. Int J Syst Evol Microbiol, 2016, 66(11):4744-4753. doi: 10.1099/ijsem.0.001421 URL
[31]	Chen Y, Chen YC, Wu JY, et al. The effect of biotic and abiotic environmental factors on Pd(II)adsorption and reduction by Bacillus wiedmannii MSM[J]. Ecotoxicol Environ Saf, 2018, 162:546-553. doi: 10.1016/j.ecoenv.2018.07.043 URL
[32]	Prudêncio de Araújo V, Lira Junior MA, et al. Bacteria from tropical semiarid temporary ponds promote maize growth under hydric stress[J]. Microbiol Res, 2020, 240:126564. doi: 10.1016/j.micres.2020.126564 URL
[33]	Glick BR. Modulation of plant ethylene levels by the bacterial enzyme ACC deaminase[J]. FEMS Microbiol Lett, 2005, 251(1):1-7. doi: 10.1016/j.femsle.2005.07.030 URL
[34]	王欢, 韩丽珍. 4株茶树根际促生菌菌株的鉴定及促生作用[J]. 微生物学通报, 2019, 46(3):548-562.
	Wang H, Han LZ. Identification of four plant growth-promoting rhizobacteria isolated from tea rhizosphere[J]. Microbiol China, 2019, 46(3):548-562.
[35]	张芬芬, 周晓伦, 贺洋洋. 一株溶磷促生菌的分离、鉴定及其对玉米幼苗生长的影响[J]. 广东农业科学, 2021, 48(5):76-82.
	Zhang FF, Zhou XL, He YY. Isolation and identification of a phosphorus-solubilizing growth-promoting bacterium and its effect on the growth of Zea mays L. seedlings[J]. Guangdong Agric Sci, 2021, 48(5):76-82.
[36]	Egamberdiyeva D. The effect of plant growth promoting bacteria on growth and nutrient uptake of maize in two different soils[J]. Appl Soil Ecol, 2007, 36(2/3):184-189. doi: 10.1016/j.apsoil.2007.02.005 URL
[37]	de Freitas JR, Germida JJ. Growth promotion of winter wheat by fluorescent pseudomonads under growth chamber conditions[J]. Soil Biol Biochem, 1992, 24(11):1127-1135. doi: 10.1016/0038-0717(92)90063-4 URL