玉米根际多功能促生菌的筛选及其对冬小麦-夏玉米轮作体系产量提升效果

doi:10.13560/j.cnki.biotech.bull.1985.2023-0690

摘要/Abstract

摘要：

【目的】黄淮海平原小麦玉米生产区主要实行冬小麦-夏玉米轮作种植模式。砂质潮土是广泛分布于黄淮海地区的土壤，属性和衍生障碍较多，包括结构性较差、蓄水保肥能力较弱等。为了提高肥料利用率、改善土壤肥力，综合实现作物产量提升和品质优化，筛选了一株多功能促生菌，并在该轮作体系验证其广谱促生效应。【方法】从玉米根际砂质潮土中筛选多功能促生菌，测定其产吲哚乙酸（IAA）、溶有机磷、解钾能力。通过形态学、生理生化及16S rDNA序列分析方法对其种属进行鉴定。摇瓶条件下探究其产IAA的最适条件，通过玉米盆栽验证其促生能力，通过冬小麦-夏玉米大田试验验证其在轮作体系中的广谱性增产效果。【结果】（1）试验筛选得到一株命名为YM3的多功能根际促生菌枯草芽孢杆菌（Bacillus subtilis），其产IAA能力达到59.21 mg/L、解有机磷能力达到0.72 mg/L、解钾能力达到18.56 mg/L。当装液量为25 mL/250 mL，pH 6-8范围内，分别以麦芽糖、蛋白胨为碳、氮源时，YM3产IAA能力最佳。（2）玉米盆栽试验结果可见，与接种灭活菌相比，接种YM3菌水剂的土壤IAA、速效磷、速效钾含量分别显著提高75.00%、48.66%、20.00%。玉米幼苗的根长、根表面积、根体积、根尖数、根分枝数分别显著增加67.95%、59.21%、51.13%、71.34%、92.06%。玉米植株的鲜重、株高、相对叶绿素含量、全氮、全磷、全钾分别显著提高了39.86%、23.51%、18.27%、17.68%、52.26%和36.53%。（3）小麦玉米轮作大田试验结果表明，接种YM3菌剂的小麦大田土壤有效氮、速效磷、速效钾分别显著增加了9.08%、13.78%、16.66%，增产率达到42.18%。玉米大田土壤速效磷和速效钾含量分别显著增加了19.18%和15.95%，增产率达到13.22%。【结论】筛选得到的枯草芽孢杆菌YM3菌株兼具产IAA、溶有机磷、解钾功能，在黄淮海平原小麦玉米轮作体系土壤中适应性强，广谱性强，能够提升砂质潮土土壤肥力，提高冬小麦-夏玉米轮作体系的产量。

关键词: 根际促生菌, IAA, 溶磷解钾, 小麦玉米轮作, 产量

Abstract:

【Objective】In China's wheat-maize production region, the Huang-Huai-Hai Plain primarily employs a winter wheat-summer maize rotation cropping system. Sandy loam soil, widely distributed in the Huang-Huai-Hai region, demonstrates various properties and associated challenges, including poor structural integrity, and weak water retention and nutrient-holding capabilities. To improve fertilizer utilization, enhance soil fertility, optimize crop yields, and improve quality, we identified a multi-functional plant growth-promoting rhizobacteria（PGPR）and validated its broad-spectrum growth-promoting effects within this rotation system. 【Method】From the sandy loam soil within the maize rhizosphere, we isolated a multi-functional PGPR bacterium and evaluated its ability of producing indole-3-acetic acid（IAA）, organic phosphorus solubilization, and potassium release ability. We identified the bacterium at the species level using morphological, physiological, biochemical, and 16S rDNA sequence analysis. Under shaken culture conditions, we determined the optimal conditions for IAA production by PGPR YM3. Subsequently, we assessed its growth-promoting ability through maize pot experiments. We verified its broad-spectrum yield-increasing effects in a field trial within the winter wheat-summer maize rotation system. 【Result】1）a multi-functional root-associated growth-promoting bacterium named YM3 was isolated, identified as Bacillus subtilis, and found to produce 59.21 mg/L IAA, solubilizing 0.72 mg/L organic phosphoruing, and releasing 18.56 mg/L potassium. The optimal conditions for IAA production by YM3 were achieved when using maltose and peptone as carbon and nitrogen sources, respectively, within a pH range of 6-8 and a liquid volume of 25 mL/250 mL. 2）Maize pot experiments revealed that soil IAA, available phosphorus, and available potassium levels significantly increased by 75.00%, 48.66%, and 20.00%, respectively, compared to soils treated with heat-inactivated YM3 bacterial suspension. Maize seedlings exhibited substantial improvements in root length, root surface area, root volume, root tip number, and root branch number, by 67.95%, 59.21%, 51.13%, 71.34%, and 92.06%, respectively. Additionally, maize plants had significant increases in fresh weight, plant height, relative chlorophyll content, total nitrogen, total phosphorus, and total potassium, with enhancements of 39.86%, 23.51%, 18.27%, 17.68%, 52.26%, and 36.53%, respectively. 3）Field trials within the winter wheat-summer maize rotation system demonstrated that soil available nitrogen, available phosphorus, and available potassium levels significantly increased by 9.08%, 13.78%, and 16.66%, respectively, with a yield increase rate of 42.18% upon YM3 bacterium agent application. Maize field soil showed notable increments of 19.18% and 15.95% in available phosphorus and available potassium, respectively, with a yield increase rate of 13.22%. 【Conclusion】The isolated B. subtilis strain YM3, possessing the ability to produce IAA, solubilize organic phosphorus, and release potassium, demonstrates strong adaptability and broad-spectrum growth-promoting effects in sandy loam soil of the Huang-Huai-Hai Plain. Moreover, it enhances soil fertility and elevates yields within the winter wheat-summer maize rotation system.

Key words: plant growth-promoting rhizobacteria, IAA, solubilizing organic phosphorus and release potassium, wheat-maize rotation, crop yield

常泸尹, 王中华, 李凤敏, 高梓源, 张辉红, 王祎, 李芳, 韩燕来, 姜瑛. 玉米根际多功能促生菌的筛选及其对冬小麦-夏玉米轮作体系产量提升效果[J]. 生物技术通报, 2024, 40(1): 231-242.

CHANG Lu-yin, WANG Zhong-hua, LI Feng-min, GAO Zi-yuan, ZHANG Hui-hong, WANG Yi, LI Fang, HAN Yan-lai, JIANG Ying. Screening Multi-functional Rhizobacteria from Maize Rhizosphere and Their Ehancing Effects on Winter Wheat-Summer Maize Rotation System[J]. Biotechnology Bulletin, 2024, 40(1): 231-242.

图/表 11

表1 供试土壤基本性质

Table 1 Basic properties of soil for testing

土壤 Soil	有机碳 Organic carbon/（g·kg^-1）	全磷 Total P/（g·kg^-1）	速效磷 Available P/（mg·kg^-1）	全钾 Total K/（g·kg^-1）	速效钾 Available K/（mg·kg^-1）	pH
砂质潮土 Sandy fluvo-aquic soil	1.91	0.29	3.44	19.56	20.42	7.39

图1 YM3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 YM3 strain

表2 YM3菌株的生理生化特性

Table 2 Physiological and biochemical characteristics of YM3 strain

项目Item	结果Results	项目Item	结果 Results
革兰氏染色 Gram stain	+	淀粉水解 Amylohydrolysis test	+
好氧性试验 Aerobic test	兼性厌氧 Facultative anaerobic	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	+
V-P试验 Voges-Proskauer test	+

图2 YM3菌株16S rDNA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 YM3 strain The scale represents 5 nucleoside substitutions per 10 000 nucleosides

图3 不同培养条件下YM3菌株的产IAA能力和生长状况（OD600）

Fig. 3 IAA-producing ability and growth status（OD600）of YM3 strain under different cultural conditions

图4 接种菌株YM3培养30 d后土壤IAA及速效钾、速效磷含量 *和**分别表示处理间在0.05和0.01水平差异显著。下同

Fig. 4 Soil IAA, available potassium, and available phosphorus contents after 30 d of inoculation with strain YM3 * and ** indicate significant difference between treatments at 0.05 and 0.01 levels, respectively. The same below

表3 接种菌株YM3对玉米幼苗根系结构和根系分级的影响

Table 3 Effects of inoculation with strain YM3 on the root system structure and root classification of maize seedlings

项目 Item	CK	YM3
总根长RL/cm	886.39±220.22	1 488.67±210.93*
各分级总根长 RL of each class/cm
I/（RD 0.0-0.5 mm）	691.11±165.95	1 192.04±157.68*
II/（RD 0.5-1.0 mm）	125.53±46.42	203.13±62.03
III/（RD 1.0-1.5 mm）	46.56±8.97	59.59±3.35
IV/（RD>1.5 mm）	22.92±0.67	33.08±5.63
根表面积RSA/cm²	121.61±27.37	193.61±16.59*
各分级根表面积 RSA of each class/cm²
I/（RD 0.0-0.5 mm）	46.43±13.62	74.04±4.67*
II/（RD 0.5-1.0 mm）	27.97±10.88	45.50±14.92
III/（RD 1.0-1.5 mm）	17.41±3.43	22.41±0.99
IV/（RD>1.5 mm）	15.49±1.85	23.97±4.47
根体积RV/cm³	1.33±0.26	2.01±0.07*
各分级根体积 RV of each class/cm³
I/（RD 0.0-0.5 mm）	14.26±3.19	23.57±1.49*
II/（RD 0.5-1.0 mm）	8.90±3.46	14.48±4.75
III/（RD 1.0-1.5 mm）	5.54±1.09	7.13±0.32
IV/（RD>1.5 mm）	4.93±0.59	7.63±1.42
根平均直径RD/mm	0.44±0.01	0.42±0.02
根尖数RT	4 505.66±1 199.46	7 720.02±1 115.10*
分枝数RF	4 760.67±780.30	9 143.33±2 714.33*

图5 接种菌株YM3对玉米幼苗生长状况和养分含量的影响

Fig. 5 Effects of inoculation with strain YM3 on the growth status and nutrient content of maize seedlings

图6 玉米幼苗及土壤中各指标之间的主成分分析

Fig. 6 Principal component analysis between maize seedlings and soil indicators

图7 玉米幼苗与土壤中指标之间的相关性分析和热图

Fig. 7 Correlation analysis and heat map among indicators in maize seedlings and soil

图8 YM3对冬小麦夏玉米土壤理化性质及产量性状的影响

Fig. 8 Effects of YM3 on the soil physicochemical properties and yield traits of wheat and corn

参考文献 37

[1]	冯倩倩, 韩惠芳, 张亚运, 等. 耕作方式对麦-玉轮作农田固碳、保水性能及产量的影响[J]. 植物营养与肥料学报, 2018, 24(4): 869-879.
	Feng QQ, Han HF, Zhang YY, et al. Effects of tillage methods on soil carbon sequestration and water holding capacity and yield in wheat-maize rotation[J]. J Plant Nutr Fertil, 2018, 24(4): 869-879.
[2]	穆文强, 康慎敏, 李平兰. 根际促生菌对植物的生长促进作用及机制研究进展[J]. 生命科学, 2022, 34(2): 118-127.
	Mu WQ, Kang SM, Li PL. Advances in rhizosphere growth-promoting bacteria function on plant growth facilitation and their mechanisms[J]. Chin Bull Life Sci, 2022, 34(2): 118-127.
[3]	Pantoja-Guerra M, Valero-Valero N, Ramírez CA. Total auxin level in the soil-plant system as a modulating factor for the effectiveness of PGPR inocula: a review[J]. Chem Biol Technol Agric, 2023, 10(1): 6. doi: 10.1186/s40538-022-00370-8
[4]	Ha-Tran DM, Nguyen TTM, Hung SH, et al. Roles of plant growth-promoting rhizobacteria(PGPR)in stimulating salinity stress defense in plants: a review[J]. Int J Mol Sci, 2021, 22(6): 3154. doi: 10.3390/ijms22063154 URL
[5]	Ali S, Khan N. Delineation of mechanistic approaches employed by plant growth promoting microorganisms for improving drought stress tolerance in plants[J]. Microbiol Res, 2021, 249: 126771. doi: 10.1016/j.micres.2021.126771 URL
[6]	Tariq M. Antagonistic features displayed by plant growth promoting rhizobacteria(PGPR): a review[J]. J Plant Sci Phytopathol, 2017, 1(1): 38-43. doi: 10.29328/journal.jpsp URL
[7]	周益帆, 白寅霜, 岳童, 等. 植物根际促生菌促生特性研究进展[J]. 微生物学通报, 2023, 50(2): 644-666.
	Zhou YF, Bai YS, Yue T, et al. Research progress on the growth-promoting characteristics of plant growth-promoting rhizobacteria[J]. Microbiol China, 2023, 50(2): 644-666.
[8]	Beneduzi A, Ambrosini A, Passaglia LMP. Plant growth-promoting rhizobacteria(PGPR): their potential as antagonists and biocontrol agents[J]. Genet Mol Biol, 2012, 35(4 suppl): 1044-1051. doi: 10.1590/S1415-47572012000600020 URL
[9]	Shabaan M, Asghar HN, Zahir ZA, et al. Salt-tolerant PGPR confer salt tolerance to maize through enhanced soil biological health, enzymatic activities, nutrient uptake and antioxidant defense[J]. Front Microbiol, 2022, 13: 901865. doi: 10.3389/fmicb.2022.901865 URL
[10]	Adedayo AA, Babalola OO, Prigent-Combaret C, et al. The application of plant growth-promoting rhizobacteria in Solanum lycopersicum production in the agricultural system: a review[J]. PeerJ, 2022, 10: e13405. doi: 10.7717/peerj.13405 URL
[11]	Libbert E, Risch H. Interactions between plants and epiphytic bacteria regarding their auxin metabolism. V. isolation and identification of the IAA-producing and destroying bacteria from pea plants[J]. Physiol Plant, 1969, 22(1): 51-58.
[12]	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
[13]	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.
[14]	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.
	Department of Microbiology, Nanjing Institute of Soil Research, Chinese Academy of Sciences. Soil microorganism research method[M]. Beijing: Science Press, 1985.
[15]	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. Handbook of identification of common bacterial systems[M]. Beijing: Science Press, 2001.
[16]	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 microscope technology and its application[M]. Xiamen, China: Xiamen University Press, 2006.
[17]	Saghai-Maroof MA, Soliman KM, Jorgensen RA, et al. Ribosomal DNA spacer-length polymorphisms in barley: Mendelian inheritance, chromosomal location, and population dynamics[J]. Proc Natl Acad Sci USA, 1984, 81(24): 8014-8018. doi: 10.1073/pnas.81.24.8014 pmid: 6096873
[18]	Jiang Y, Wu Y, Hu N, et al. Interactions of bacterial-feeding nematodes and indole-3-acetic acid(IAA)-producing bacteria promotes growth of Arabidopsis thaliana by regulating soil auxin status[J]. Appl Soil Ecol, 2020, 147: 103447. doi: 10.1016/j.apsoil.2019.103447 URL
[19]	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. Beijing: China Agriculture Press, 2000.
[20]	Shi GR, Xia SL, Ye J, et al. PEG-simulated drought stress decreases cadmium accumulation in castor bean by altering root morphology[J]. Environ Exp Bot, 2015, 111: 127-134. doi: 10.1016/j.envexpbot.2014.11.008 URL
[21]	Bhattacharyya PN, Jha DK. Plant growth-promoting rhizobacteria(PGPR): emergence in agriculture[J]. World J Microbiol Biotechnol, 2012, 28(4): 1327-1350. doi: 10.1007/s11274-011-0979-9 URL
[22]	Ma SY, Lin YB, Qin YQ, et al. Microbial diversity characteristics of Areca palm rhizosphere soil at different growth stages[J]. Plants, 2021, 10(12): 2706. doi: 10.3390/plants10122706 URL
[23]	Ullah A, Akbar A, Luo QQ, et al. Microbiome diversity in cotton rhizosphere under normal and drought conditions[J]. Microb Ecol, 2019, 77(2): 429-439. doi: 10.1007/s00248-018-1260-7 pmid: 30196314
[24]	de O Nunes PS, de Oliveira TS, et al. Bacillus subtilis and Bacillus licheniformis promote tomato growth[J]. Publ Braz Soc Microbiol, 2023, 54(1): 397-406.
[25]	王舒, 张林平, 郝菲菲, 等. 油茶根际高效溶磷细菌的筛选、鉴定及其安全性测试[J]. 林业科学研究, 2015, 28(2): 166-172.
	Wang S, Zhang LP, Hao FF, et al. Screening, identification and security test of Camellia oleifera rhizosphere phosphate-solubilizing bacteria[J]. For Res, 2015, 28(2): 166-172.
[26]	徐文思, 姜瑛, 李引, 等. 一株植物促生菌的筛选、鉴定及其对花生的促生效应研究[J]. 土壤, 2014, 46(1): 119-125.
	Xu WS, Jiang Y, Li Y, et al. Isolation, identification of plant growth-promoting bacteria and its promoting effects on peanuts[J]. Soils, 2014, 46(1): 119-125.
[27]	Liang CY, Tian J, Liao H. Proteomics dissection of plant responses to mineral nutrient deficiency[J]. Proteomics, 2013, 13(3/4): 624-636. doi: 10.1002/pmic.v13.3-4 URL
[28]	Keswani C, Singh SP, Cueto L, et al. Auxins of microbial origin and their use in agriculture[J]. Appl Microbiol Biotechnol, 2020, 104(20): 8549-8565. doi: 10.1007/s00253-020-10890-8 pmid: 32918584
[29]	Niu YF, Jin GL, Li X, et al. Phosphorus and magnesium interactively modulate the elongation and directional growth of primary roots in Arabidopsis thaliana(L.) Heynh[J]. J Exp Bot, 2015, 66(13): 3841-3854. doi: 10.1093/jxb/erv181 URL
[30]	Tian XL, Wang GW, Zhu R, et al. Conditions and indicators for screening cotton(Gossypium hirsutum L.) varieties tolerant to low potassium[J]. Acta Agron Sin, 2008, 34(8): 1435-1443.
[31]	He AL, Zhao LY, Ren W, et al. A volatile producing Bacillus subtilis strain from the rhizosphere of Haloxylon ammodendron promotes plant root development[J]. Plant Soil, 2023, 486(1): 661-680. doi: 10.1007/s11104-023-05901-2
[32]	勾宇春, 王宗抗, 张志鹏, 等. 植物根际促生菌作用机制研究进展[J]. 应用与环境生物学报, 2023, 29(2): 495-506.
	Gou YC, Wang ZK, Zhang ZP, et al. Advance in role mechanisms of plant growth-promoting rhizobacteria[J]. Chin J Appl Environ Biol, 2023, 29(2): 495-506.
[33]	李刚强, 王楠, 李永斌, 等. 两种固氮芽孢杆菌菌剂在小麦—玉米轮作区大田试验效果评价[J]. 中国农业科技导报, 2020, 22(4): 147-152. doi: 10.13304/j.nykjdb.2019.0556
	Li GQ, Wang N, Li YB, et al. Effect evaluation of field experiment of two Paenibacillus sp. agents in wheat-maize rotation area[J]. J Agric Sci Technol, 2020, 22(4): 147-152.
[34]	Sapre S, Gontia-Mishra I, Tiwari S. Klebsiella sp. confers enhanced tolerance to salinity and plant growth promotion in oat seedlings(Avena sativa)[J]. Microbiol Res, 2018, 206: 25-32. doi: 10.1016/j.micres.2017.09.009 URL
[35]	Elsharkawy MM, Elsawy MM, Ismail IA. Mechanism of resistance to Cucumber mosaic virus elicited by inoculation with Bacillus subtilis subsp. subtilis[J]. Pest Manag Sci, 2022, 78(1): 86-94. doi: 10.1002/ps.v78.1 URL
[36]	Xie DS, Cai XW, Yang CP, et al. Studies on the control effect of Bacillus subtilis on wheat powdery mildew[J]. Pest Manag Sci, 2021, 77(10): 4375-4382. doi: 10.1002/ps.v77.10 URL
[37]	Meena M, Swapnil P, Divyanshu K, et al. PGPR-mediated induction of systemic resistance and physiochemical alterations in plants against the pathogens: current perspectives[J]. J Basic Microbiol, 2020, 60(10): 828-861. doi: 10.1002/jobm.v60.10 URL