耐盐促生芽孢杆菌的筛选及其对盐胁迫下燕麦生长的影响

doi:10.3969/j.issn.1004-1524.2022.06.17

浙江农业学报 ›› 2022, Vol. 34 ›› Issue (6): 1268-1276.DOI: 10.3969/j.issn.1004-1524.2022.06.17

耐盐促生芽孢杆菌的筛选及其对盐胁迫下燕麦生长的影响

李丽艳¹^,²(), 谭海霞³^,^*(), 李婧¹^,², 王连龙³, 杜迎辉¹^,², 徐志文¹

1.领先生物农业股份有限公司,河北秦皇岛 066000
2.河北省农业生物技术技术创新中心,河北秦皇岛 066000
3.河北环境工程学院生态学系,河北秦皇岛 066000

收稿日期:2021-07-09 出版日期:2022-06-25 发布日期:2022-06-30
作者简介:*谭海霞,E-mail： tanhaixia2001@126.com
李丽艳（1985—）,女,河北唐山人,硕士,农艺师,研究方向为微生物肥料研发与应用。E-mail： li.liyan@leadst.cn
通讯作者: 谭海霞
基金资助:
国家自然科学基金(30870419);河北省重点研发计划(1927302D);河北环境工程学院青年基金(2018ZRQN02);秦皇岛市市级科学技术研究与发展计划(202005A036)

Screening of salt-tolerant growth-promoting Bacillus strains and their effect on oat growth under salt stress

LI Liyan¹^,²(), TAN Haixia³^,^*(), LI Jing¹^,², WANG Lianlong³, DU Yinghui¹^,², XU Zhiwen¹

1. Leading Biological and Agricultural Limited Company, Qinhuangdao 066000, Hebei, China
2. Hebei Agricultural Biotechnology Technology Innovation Center, Qinhuangdao 066000, Hebei, China
3. Department of Ecology, Hebei University of Environmental Engineering, Qinhuangdao 066000, Hebei, China

Received:2021-07-09 Online:2022-06-25 Published:2022-06-30
Contact: TAN Haixia

摘要/Abstract

摘要：

从河北省秦皇岛滨海盐生植物根际土壤分离筛选耐盐促生芽孢杆菌,研究其在盐胁迫条件下对燕麦生长的促生效果,以期为研发耐盐促生菌剂和菌肥提供菌种资源。采用pH值9.0和NaCl质量分数分别为5%、10%、15%的LB培养基筛选、分离耐高盐的芽孢杆菌菌株,用功能培养基从中筛选具有促生能力的细菌菌株,用Salkowski比色法定性定量分析其产IAA的能力,采用盆栽试验研究其在盐胁迫条件下对燕麦生长的影响,运用16S rDNA序列分析法对促生效果好的菌株进行鉴定。结果显示,本研究共分离得到13株耐高盐的芽孢杆菌菌株,其中3株芽孢杆菌（YP2、YP4、SM12）可耐受10%（质量分数）的NaCl,且均有解磷、解钾、固氮能力,具有较强的产IAA能力。接种这3株菌株均能在盐胁迫条件下促进燕麦生长,提高其抗盐能力,其中菌株YP2的效果最优,与对照相比,其株高、茎粗、地上部鲜重、总根长、根系总表面积、根尖数分别显著（P<0.05）增加72.02%、42.58%、186.11%、392.35%、378.07%和518.85%,燕麦叶片中的丙二醛含量显著（P<0.05）降低43.34%,叶绿素含量、脯氨酸含量,及过氧化物酶、过氧化氢酶活性分别显著（P<0.05）提高了312.20%、124.10%、274.09%和198.60%。经16S rDNA序列分析,将菌株YP2初步鉴定为弯曲芽孢杆菌（Bacillus flexus）。该菌株作为盐碱地专用生物菌剂具有较大的开发应用潜力。

关键词: 耐盐促生菌, 芽孢杆菌, 盐胁迫, 生理特性, 燕麦

Abstract:

Abstract: In order to screen salt-tolerant growth-promoting Bacillus strains from the rhizosphere soil of coastal halophytes in Qinhuangdao City, Hebei Province, and provide bacterial resources for the development of salt-tolerant bacterial manure, high-salinity tolerant Bacillus strains were screened and separated by LB culture medium with pH of 9.0 and NaCl mass fraction of 5%, 10%, 15%, respectively. The strains with growth-promoting abilities were further screened by functional medium. Their auxin (IAA)-producing abilities were quantitatively analyzed by Salkowski colorimetric method. The pot experiment was used to study their effect on the growth of oat under salt stress, and 16S rDNA sequence analysis was used to identify the strain with good growth promoting effect. It was shown that a total of 13 Bacillus strains were isolated with high salt tolerance, among which 3 strains (YP2, YP4, SM12) could tolerant 10% (mass fraction) NaCl. All the three strains had abilities of phosphate-solubilizing, potassium-releasing, nitrogen fixation, and production of IAA. Besides, all the three strains could improve the salt resistance of oat and promote its growth under salt stress, and the strain YP2 showed the best performance. Compared with the control, the plant height, stem diameter, shoot biomass, root length, root surface area and root tips quantity were significantly (P<0.05) increased by 72.02%, 42.58%, 186.11%, 392.35%, 378.07% and 518.85%, respectively, malondialdehyde content in oat leaves was significantly (P<0.05) decreased by 43.34%, and the contents of chlorophyll and proline, and activities of peroxidase and catalase were significantly (P<0.05) increased by 312.20%, 124.10%, 274.09% and 198.60%, respectively. As revealed by 16S rDNA sequences, YP2 strain was identified as Bacillus flexus, which has great potential to be developed as a special bacterial agent.

Key words: salt tolerant growth-promoting bacteria, Bacillus, salt stress, physiological characteristics, oat

中图分类号:

S144

李丽艳, 谭海霞, 李婧, 王连龙, 杜迎辉, 徐志文. 耐盐促生芽孢杆菌的筛选及其对盐胁迫下燕麦生长的影响[J]. 浙江农业学报, 2022, 34(6): 1268-1276.

LI Liyan, TAN Haixia, LI Jing, WANG Lianlong, DU Yinghui, XU Zhiwen. Screening of salt-tolerant growth-promoting Bacillus strains and their effect on oat growth under salt stress[J]. Acta Agriculturae Zhejiangensis, 2022, 34(6): 1268-1276.

图/表 9

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盐生植物 Halophyte	pH	碱解氮 Alkaline N/ (mg·kg^-1)	有效磷 Available P/ (mg·kg^-1)	速效钾 Available K/ (mg·kg^-1)	有机质 Organic matter/ (g·kg^-1)	水溶性盐分 Water soluble salt/( g·kg^-1)
盐地碱蓬 Suaeda salsa	8.55	19	10.3	256	4.69	4.0
盐角草 Salicornia europaea	8.57	47	18.4	286	7.20	5.0
芦苇 Phragmites australis	8.69	30	15.7	197	7.04	4.1
水麦冬 Triglochin palustre	8.03	27	7.7	274	5.10	4.5

盐生植物 Halophyte	pH	碱解氮 Alkaline N/ (mg·kg^-1)	有效磷 Available P/ (mg·kg^-1)	速效钾 Available K/ (mg·kg^-1)	有机质 Organic matter/ (g·kg^-1)	水溶性盐分 Water soluble salt/( g·kg^-1)
盐地碱蓬 Suaeda salsa	8.55	19	10.3	256	4.69	4.0
盐角草 Salicornia europaea	8.57	47	18.4	286	7.20	5.0
芦苇 Phragmites australis	8.69	30	15.7	197	7.04	4.1
水麦冬 Triglochin palustre	8.03	27	7.7	274	5.10	4.5

菌株来源 Sampling site	NaCl质量分数 NaCl mass fraction/%	分离菌株数 Isolates quantity	菌株编号 Strain No.
盐地碱蓬 Suaeda salsa	5	3	YP1~YP3
	10	2	YP4~YP5
	15	1	YP6
盐角草 Salicornia europaea	5	1	YJ7
盐角草 Salicornia europaea	10	1	YJ8
芦苇 Phragmites australis	5	2	LW9~LW10
芦苇 Phragmites australis	10	1	LW11
水麦冬 Triglochin palustre	5	2	SM12~SM13

菌株来源 Sampling site	NaCl质量分数 NaCl mass fraction/%	分离菌株数 Isolates quantity	菌株编号 Strain No.
盐地碱蓬 Suaeda salsa	5	3	YP1~YP3
	10	2	YP4~YP5
	15	1	YP6
盐角草 Salicornia europaea	5	1	YJ7
盐角草 Salicornia europaea	10	1	YJ8
芦苇 Phragmites australis	5	2	LW9~LW10
芦苇 Phragmites australis	10	1	LW11
水麦冬 Triglochin palustre	5	2	SM12~SM13

菌株编号 Strain No.	IAA产生活性 IAA production activity	溶解有机磷 Organic phosphate solubilization	溶解无机磷 Inorganic phosphate solubilization	解钾 Potassium-releasing	固氮 Nitrogen fixation	IAA产生量 IAA yield/(mg·L^-1）
YP1	+	—	+	+	—	25.76
YP2	+++	++	++	++	++	62.10
YP3	+	—	—	+	+	33.27
YP4	+++	—	++	++	+	50.00
YP5	++	+	+	++	+	40.30
YP6	++	+	—	—	+	36.55
YJ7	+	—	—	—	+	28.19
YJ8	+	—	+	—	+	25.46
LW9	+	—	+	—	+	20.32
LW10	+	+	+	+	+	18.57
LW11	+	+	+	—	—	10.11
SM12	++	+	—	+	++	43.80
SM13	+	—	—	+	+	29.57

耐盐促生芽孢杆菌的筛选及其对盐胁迫下燕麦生长的影响

Screening of salt-tolerant growth-promoting Bacillus strains and their effect on oat growth under salt stress

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菌株编号 Strain No.	pH								NaCl质量分数NaCl mass fraction/%
菌株编号 Strain No.	3.0	4.0	5.0	6.0	7.0	8.0	9.0	10.0	0.5	1	5	10	15
YP2	—	—	++	+++	+++	+++	+++	+	+++	+++	+++	++	+
YP4	—	—	+	+++	+++	+++	++	—	+++	+++	+++	+	—
SM12	—	—	—	+++	+++	+++	++	—	+++	+++	++	+	—

处理 Treatment	株高 Plant height/cm	茎粗 Stem diameter/mm	地上部鲜重 Shoot biomass/g	总根长 Root length/cm	根系总表面积 Root surface area/cm²	根系直径 Root. diameter/mm	根尖数 Root tips quantity
CK	37.21±7.75 c	2.09±0.21 b	1.44±0.50 c	76.5±13.3 c	32.33±8.35 c	1.35±0.15 a	62.12±7.3 c
YP2	64.01±8.26 a	2.98±0.33 a	4.12±1.24 a	376.5±38.3 a	154.56±31.25 a	1.37±0.11 a	384.43±13.16 a
YP4	55.17±8.83 b	2.25±0.38 b	2.59±1.55 b	228.2±38.0 b	90.43±24.87 b	1.22±0.10 a	247.83±14.28 b
SM12	62.66±9.41 ab	2.68±0.25 a	3.44±0.85 ab	277.3±37.1 b	102.97±13.09 b	1.26±0.12 a	256.34±18.14 b

处理 Treatment	PC1得分 Score of PC1	PC2得分 Score of PC2	综合得分 Total score	排名 Rank
CK	-1.365	0.621	-0.866	4
YP2	1.018	1.010	1.016	1
YP4	0.031	-1.177	-0.156	3
SM12	0.316	-0.454	0.122	2

处理 Treatment	叶绿素 Chlorophyll/(mg·g^-1)	丙二醛Malondialdehyde/ (μmol·g^-1)	脯氨酸 Proline/(μg·g^-1)	过氧化物酶 Peroxidase/(U·g^-1·min^-1）	过氧化氢酶Catalase/(mg·g^-1·min^-1）
CK	0.82±0.03 c	15.02±0.16 a	0.83±0.04 c	163.33±8.72 c	12.13±0.21 d
YP2	3.38±0.07 a	8.51±0.44 d	1.86±0.04 a	611.00±3.21 a	36.22±0.60 a
YP4	0.91±0.12 bc	13.58±0.33 b	1.64±0.06 b	483.67±4.16 b	15.98±0.41 c
SM12	1.00±0.08 b	12.13±0.30 c	1.68±0.05 b	499.50±4.58 b	31.28±0.36 b

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