蓝莓内生细菌的分离及其对上海青铝胁迫的缓解作用

doi:10.13560/j.cnki.biotech.bull.1985.2024-0978

摘要/Abstract

摘要：

目的分离蓝莓内生细菌并探究其对上海青生长铝胁迫的缓解作用，为提高植物抗酸耐铝能力提供菌种资源。方法采用组织块培养法分离蓝莓叶芽内生细菌，经划线纯化后液体培养测定其耐铝特性。通过上海青水培实验，测定上海青生物量、叶绿素、可溶性蛋白、可溶性糖含量，SOD、POD、CAT活性及培养液电导率等指标。结合形态学特征、生理生化特征、16S rDNA及gryB基因组测序对功能优异菌株进行鉴定。采用溶血试验验证菌株毒性。结果自蓝莓叶芽内分离得到3株内生细菌（Y01、Y02、Y03），其中，Y01、Y03菌株在低于1 000 μmol/L铝离子条件下均表现出良好的耐受性。水培条件下，上海青在100 μmol/L铝浓度时长势最佳，但300 μmol/L铝对上海青的生长产生严重胁迫，生物量均明显降低。3株内生细菌均可不同程度缓解铝毒，其中Y01菌株效果最佳，经其处理后，上海青株高、根长、鲜重及叶绿素含量分别增加95.6%、40.2%、594.7%、22.9%。上海青叶片SOD、POD、CAT活性、可溶性糖与可溶性蛋白含量分别显著升高30.8%、7.8%、78.3%、91.7%、202.5%；根系SOD、POD、CAT活性、可溶性糖与可溶性蛋白含量分别显著升高118.2%、101.2%、59.6%、79.2%、74.3%，并维持在较高水平。经鉴定Y01菌株为蜡样芽胞杆菌（Bacillus cereus），该菌株溶血试验呈阳性。结论蓝莓内生细菌Y01通过提高叶绿素含量、抗氧化酶活性及可溶性物质的释放等途径，缓解铝胁迫对上海青植株细胞膜等器官的毒害作用。该菌株可为提高植物抗酸耐铝能力提供新的菌种资源。

关键词: 铝胁迫, 内生细菌, 上海青, 耐铝机制, 细胞膜透性, 抗氧化酶活性

Abstract:

Objective Isolation of endophytic bacteria from blueberry and investigation of their mitigating effects on aluminum stress in Brassica chinensis L. growth are aimed to provide strain resources for improving the acid and aluminum tolerance capacity of plants. Method Tissue block culture method was used to isolate the endophytic bacteria from blueberry leaf buds, and determine their aluminum tolerance characteristics by shaker culture. The dominant strains were identified by combining morphological characteristics, physiological and biochemical characteristics, 16S rDNA and gryB genome sequencing. The toxicity of the strains was verified by hemolysis test. Result Three endophytic bacteria (Y01, Y02, and Y03) were isolated from the leaf buds of blueberry, strains Y01 and Y03 showed fine tolerance to of <1 000 μmol/L aluminum concentration. Under hydroponic conditions, Brassica chinensis L. grew best at 100 μmol/L aluminum concentration, but 300 μmol/L aluminum concentration caused severe impact on B. chinensis L., and the biomass significantly reduced. Three strains of endophytic bacteria alleviated the aluminum toxicity to varying degrees, and the best effect was achieved by strain Y01, which increased the height, root length, fresh weight and chlorophyll content of B. chinensis L. by 95.6%, 40.2%, 594.7%, 22.2%. The activities of SOD, POD and CAT, and the contents of soluble sugar and soluble protein in the leaves of B. chinensis L. significantly increased by 30.8%, 7.8%, 78.3%, 91.7% and 202.5%, respectively. The activity of SOD, POD and CAT and that content of soluble sugar and soluble protein in root system increased by 118.2%, 101.2%, 59.6%, 79.2% and 74.3%, respectively,and maintained at a high level. Strain Y01 was identified as Bacillus cereus, and the hemolytic test was positive for this strain. Conclusion Endophytic bacteria Y01 isolated from blueberry alleviates the toxic effects of aluminum stress on cell membranes and other organs of B. chinensis L. by increasing chlorophyll content, antioxidant enzyme activities and release of soluble substances. This strain may improve the acid and aluminum resistance of plants as a new strain resource.

Key words: aluminum stress, endophytic bacteria, Brassica chinensis L., aluminum-resistance mechanism, cell membrane permeability, antioxidant enzyme activity

佟德利, 张馨, 陈佳庆, 贺海升. 蓝莓内生细菌的分离及其对上海青铝胁迫的缓解作用[J]. 生物技术通报, 2025, 41(4): 302-311.

TONG De-li, ZHANG Xin, CHEN Jia-qing, HE Hai-sheng. Isolation of Endophytic Bacteria from Blueberry and Its Alleviative Effects on Brassica chinensis L. under Aluminum Stress[J]. Biotechnology Bulletin, 2025, 41(4): 302-311.

图/表 9

图1 不同铝离子浓度下细菌生长状况（OD600）Y01、Y02、Y03：蓝莓叶芽中分离得到的3株优势细菌；不同字母表示显著性差异（P<0.05）。下同

Fig. 1 Bacterial growth at different concentrations of aluminum ions (OD600)Y01, Y02, Y03: Three dominant bacterial strains isolated from blueberry leaf buds. Different letters indicate significant difference (P<0.05). The same below

图2 五种铝浓度下不同菌株对上海青生长的影响Al0‒Al300：上海青水培液铝离子浓度0‒300 μmol/L

Fig. 2 Effects of different strains on the growths of Brassica chinensis L. at five aluminium concentrationsAl0‒Al300: Aluminum ion concentration in Brassica chinensis L. hydroponic solution 0‒300 μmol/L

图3 五种铝浓度下不同菌株对生物量及叶绿素含量的影响

Fig. 3 Effects of different strains on biomass and chlorophyll contents at five aluminum concentrations

图4 五种铝浓度下不同菌株对电导率的影响

Fig. 4 Effects of different strains on conductivity at five aluminium concentrations

图5 Y01菌株对上海青叶片与根系抗氧化酶活性的影响A‒C：上海青叶片；D‒F：上海青根系；*表示P<0.05； **表示P<0.01；下同

Fig. 5 Effects of strain Y01 on the antioxidant enzyme activities of leaves and roots of B. chinensis L.A‒C: B. chinensis L. leaves. D‒F: B. chinensis L. roots. * indicates P<0.05; ** indicates P<0.01. The same below

图6 Y01菌株对上海青叶片与根系可溶性糖和可溶性蛋白的影响A、C：上海青叶片；B、D：上海青根系

Fig. 6 Effects of strain Y01 on the soluble sugars and soluble proteins of leaves and roots of B. chinensis L.A, C: B. chinensis L. leaves. B, D: B. chinensis L. roots

图7 菌株的形态学观察A、B、C为Y01、Y02、Y03菌落形态；D、E、F为Y01、Y02、Y03显微镜下革兰氏染色情况

Fig. 7 Morphological observations of the strainA, B, and C are the morphologies of Y01, Y02, and Y03 colonies; D, E, and F are the Gram staining of Y01, Y02, and Y03 under microscope

表1 三株蓝莓内生细菌部分生理生化特征

Table 1 Selected physiological and biochemical characteristics of three strains of blueberry endophytic bacteria

生理生化反应 Physiological and biochemical reaction	Y01	Y02	Y03
淀粉水解 Starch hydrolysis	+	+	+
明胶水解 Gelatin hydrolysis	+	+	+
葡萄糖水解 Glucose hydrolysis	+	-	+
接触酶活性 Contactase activity	+	+	+
V-P 反应 The V-P reaction	+	-	-
溶血试验 Hemolysis test	+	/	/

图8 基于16S rDNA序列与gyrB基因序列的Y01菌株多基因系统发育树

Fig. 8 Multigene phylogenetic tree of strain Y01 based on 16S rDNA sequence and gyrB gene sequence

参考文献 36

1	Wang Y, Yao ZS, Zhan Y, et al. Potential benefits of liming to acid soils on climate change mitigation and food security [J]. Glob Chang Biol, 2021, 27(12): 2807-2821.
2	Xing W, Lu XM, Ying JY, et al. Disentangling the effects of nitrogen availability and soil acidification on microbial taxa and soil carbon dynamics in natural grasslands [J]. Soil Biol Biochem, 2022, 164: 108495.
3	Zhu QC, Liu XJ, Hao TX, et al. Cropland acidification increases risk of yield losses and food insecurity in China [J]. Environ Pollut, 2020, 256: 113145.
4	徐仁扣, 李九玉, 周世伟, 等. 我国农田土壤酸化调控的科学问题与技术措施 [J]. 中国科学院院刊, 2018, 33(2): 160-167.
	Xu RK, Li JY, Zhou SW, et al. Scientific issues and controlling strategies of soil acidification of croplands in China [J]. Bull Chin Acad Sci, 2018, 33(2): 160-167.
5	张雅茹, 闫磊, 程锦, 等. 硼对铝胁迫下油菜幼苗生长及生理特性的影响 [J]. 中国油料作物学报, 2023, 45(1): 138-147.
	Zhang YR, Yan L, Cheng J, et al. Effects of boron on growth and physiological characteristics of rape seedlings under aluminum stress [J]. Chin J Oil Crop Sci, 2023, 45(1): 138-147.
6	李颖, 万一兵, 范又维, 等. 铝离子对植物生长发育的影响及耐铝机制研究进展 [J/OL]. 分子植物育种, 2023. .
	Li Y, Wan YB, Fan YW, et al. Research progress on the effect of Al³⁺ on plant growth and development and the mechanism of aluminum tolerance [J/OL]. Mol Plant Breed, 2023. .
7	张冉, 韩博, 任健, 等. 铝对植物毒害及草本植物耐铝毒机制研究进展 [J]. 云南农业大学学报: 自然科学, 2020, 35(2): 353-360.
	Zhang R, Han B, Ren J, et al. Research progress on aluminum toxicity to plants and mechanisms of aluminum tolerance in herbaceous [J]. J Yunnan Agric Univ Nat Sci, 2020, 35(2): 353-360.
8	范伟, 娄和强, 龚育龙, 等. 调控铝诱导根尖有机酸分泌的分子机制 [J]. 植物生理学报, 2014, 50(10): 1469-1478.
	Fan W, Lou HQ, Gong YL, et al. Molecular mechanisms regulating aluminum-induced secretion of organic acids in root apex [J]. Plant Physiol J, 2014, 50(10): 1469-1478.
9	Sasidharan R, Voesenek LA, Pierik R. Cell wall modifying proteins mediate plant acclimatization to biotic and abiotic stresses [J]. Crit Rev Plant Sci, 2011, 30(6): 548-562.
10	胡现荣, 范存敏, 吴萍萍, 等. 石灰用量对酸化稻田土壤酸度和水稻产量的影响 [J]. 安徽农业科学, 2023, 51(8): 22-25.
	Hu XR, Fan CM, Wu PP, et al. Effects of lime dosage on acidification of paddy soil and rice yield [J]. J Anhui Agric Sci, 2023, 51(8): 22-25.
11	闫静, 时仁勇, 王昌军, 等. 不同改良剂对酸性烟田的改良效果及其对烤烟生长的影响 [J]. 土壤, 2023, 55(3): 612-618.
	Yan J, Shi RY, Wang CJ, et al. Effects of different amendments on soil acidity and tobacco growth in acidic tobacco field [J]. Soils, 2023, 55(3): 612-618.
12	曾廷廷, 蔡泽江, 王小利, 等. 酸性土壤施用石灰提高作物产量的整合分析 [J]. 中国农业科学, 2017, 50(13): 2519-2527.
	Zeng TT, Cai ZJ, Wang XL, et al. Integrated analysis of liming for increasing crop yield in acidic soils [J]. Sci Agric Sin, 2017, 50(13): 2519-2527.
13	李培培, 仝昊天, 韩燕来, 等. 秸秆直接还田与炭化还田对潮土硝化微生物的影响 [J]. 土壤学报, 2019, 56(6): 1471-1481.
	Li PP, Tong HT, Han YL, et al. Effect of straw return, directly or as biochar, on nitrifying microbes in fluvo-aquic soil [J]. Acta Pedol Sin, 2019, 56(6): 1471-1481.
14	Shahzad A, Siddique A, Ferdous S, et al. Heavy metals mitigation and growth promoting effect of endophytic Agrococcus terreus (MW 979614) in maize plants under zinc and nickel contaminated soil [J]. Front Microbiol, 2023, 14: 1255921.
15	Jiang XL, Li WW, Han ML, et al. Aluminum-tolerant, growth-promoting endophytic bacteria as contributors in promoting tea plant growth and alleviating aluminum stress [J]. Tree Physiol, 2022, 42(5): 1043-1058.
16	裴彤, 周福军, 刘梓强, 等. 外源水杨酸和褪黑素对铝胁迫下蓝莓的缓解作用[J/OL].吉林农业大学学报, 2024. .
	Pei T, Zhou FJ, Liu ZQ, et al. Study on the alleviative effect of exogenous salicylic acid and melatonin on blueberry under aluminum stress[J/OL]. J Jilin Agric Univ, 2024. .
17	张盈川, 吴晓明玉, 陶保龙, 等. Bna-miR43介导甘蓝型油菜响应干旱胁迫 [J]. 植物学报, 2023, 58(5): 701-711.
	Zhang YC, Wu XMY, Tao BL, et al. Bna-miR43 mediates the response of drought tolerance in Brassica napus [J]. Chin Bull Bot, 2023, 58(5): 701-711.
18	汤绍虎, 罗充. 植物生理学实验教程 [M]. 重庆: 西南师范大学出版社, 2012.
	Tang SH, Luo C. Experimental course of plant physiology [M]. Chongqing: Southwest China Normal University Press, 2012.
19	张腾国, 胡馨丹, 李萍, 等. 盐及低温胁迫对油菜ROS和抗氧化酶活性的影响 [J]. 兰州大学学报: 自然科学版, 2019, 55(4): 497-505.
	Zhang TG, Hu XD, Li P, et al. Effects of low temperature and salt stress on the activity of ROS and antioxidant enzymes in Brassica campestris seedlings [J]. J Lanzhou Univ Nat Sci, 2019, 55(4): 497-505.
20	王晶英, 敖红, 张杰, 等. 植物生理生化实验技术与原理 [M]. 哈尔滨: 东北林业大学出版社, 2003.
	Wang JY, Ao H, Zhang J, et al. Technology and principle of plant physiology and biochemistry experiment [M]. Harbin: Northeast Forestry University Press, 2003.
21	郜欢欢, 叶桑, 王倩, 等. 甘蓝型油菜种子萌发期耐铝毒特性综合评价及其种质筛选 [J]. 作物学报, 2019, 45(9): 1416-1430.
	Gao HH, Ye S, Wang Q, et al. Screening and comprehensive evaluation of aluminum-toxicity tolerance during seed germination in Brassca napus [J]. Acta Agron Sin, 2019, 45(9): 1416-1430.
22	韩德鹏, 刘星月, 王馨悦, 等. 铝胁迫对油菜根系形态和生理指标的影响 [J]. 核农学报, 2019, 33(9): 1824-1832.
	Han DP, Liu XY, Wang XY, et al. Effects of aluminum stress on morphology parameters of roots and physiological indexes in Brassica napus L [J]. J Nucl Agric Sci, 2019, 33(9): 1824-1832.
23	邓晓霞, 李月明, 姚堃姝, 等. 植物适应酸铝胁迫机理的研究进展 [J]. 生物工程学报, 2022, 38(8): 2754-2766.
	Deng XX, Li YM, Yao KS, et al. Advances in the mechanism of plant adaptation to acid aluminum stress [J]. Chin J Biotechnol, 2022, 38(8): 2754-2766.
24	陈泰林, 钱春梅, 张建军, 等. 植物铝胁迫响应机制的研究进展 [J]. 热带农业科学, 2010, 30(2): 37-48, 86.
	Chen TL, Qian CM, Zhang JJ, et al. Advances of mechanism of plant response to aluminum stress [J]. Chin J Trop Agric, 2010, 30(2): 37-48, 86.
25	于天一, 孙秀山, 石程仁, 等. 土壤酸化危害及防治技术研究进展 [J]. 生态学杂志, 2014, 33(11): 3137-3143.
	Yu TY, Sun XS, Shi CR, et al. Advances in soil acidification hazards and control techniques [J]. Chin J Ecol, 2014, 33(11): 3137-3143.
26	吴林坤, 林向民, 林文雄. 根系分泌物介导下植物-土壤-微生物互作关系研究进展与展望 [J]. 植物生态学报, 2014, 38(3): 298-310.
	Wu LK, Lin XM, Lin WX. Advances and perspective in research on plant-soil-microbe interactions mediated by root exudates [J]. Chin J Plant Ecol, 2014, 38(3): 298-310.
27	Ali H, Khan E. What are heavy metals? Long-standing controversy over the scientific use of the term 'heavy metals'-proposal of a comprehensive definition [J]. Toxicol Environ Chem, 2018, 100(1): 6-19.
28	赵会娥, 贺立源, 章爱群, 等. 铝胁迫对植物光合作用的影响及其机理的研究进展 [J]. 华中农业大学学报, 2008, 27(1): 155-160.
	Zhao HE, He LY, Zhang AQ, et al. Advance in the study of effects of aluminum stress on plant photosynthesis and its mechanism [J]. J Huazhong Agric Univ, 2008, 27(1): 155-160.
29	Zhang J, He L, Guo C, et al. Evaluation of Aspergillus aculeatus GC-09 for the biological control of citrus blue mold caused by Penicillium italicum [J]. Fungal Biol, 2022, 126(3): 201-212.
30	符京燕, 梁林林, 周敏, 等. 伽马氨基丁酸浸种对铝胁迫下白三叶种子萌发及耐铝性的影响 [J]. 草地学报, 2020, 28(5): 1275-1284.
	Fu JY, Liang LL, Zhou M, et al. Effects of seeds soaking with γ-aminobutyric acid on germination and aluminium tolerance of white clover [J]. Acta Agrestia Sin, 2020, 28(5): 1275-1284.
31	田茂燕, 向婷颖, 钟川, 等. 番茄砧木耐热性评价及热胁迫生理响应研究 [J]. 中国蔬菜, 2021(5): 39-47.
	Tian MY, Xiang TY, Zhong C, et al. Evaluation of tomato rootstock heat resistance and studies on its physiological response to heat stress [J]. China Veg, 2021(5): 39-47.
32	张雯, 黄益丹, 张鹏超, 等. 甘蓝型油菜耐铝极端品种筛选及耐铝生理机制初步解析 [J]. 华中农业大学学报, 2023, 42(6): 154-163.
	Zhang W, Huang YD, Zhang PC, et al. Screening extreme varieties with aluminum tolerance and analyzing physiological mechanisms of aluminum tolerance in Brassica napus [J]. J Huazhong Agric Univ, 2023, 42(6): 154-163.
33	刘强, 胡华良, 苏柳, 等. 不同有机物料对铝毒土壤上油菜生长的缓解效应研究 [J]. 井冈山大学学报: 自然科学版, 2010, 31(2): 22-25.
	Liu Q, Hu HL, Su L, et al. Alleviation effect of different organic manures on rape seedings grown in aluminum toxicity soil [J]. J Jinggangshan Univ Nat Sci, 2010, 31(2): 22-25.
34	刘燕, 罗文敏, 段如雁, 等. 威宁地区茶树种质资源的抗旱性研究 [J]. 西南农业学报, 2022, 35(3): 530-535.
	Liu Y, Luo WM, Duan RY, et al. Study on drought-resistant tea cultivars in Weining county [J]. Southwest China J Agric Sci, 2022, 35(3): 530-535.
35	Plett JM, Plett KL, Wong-Bajracharya J, et al. Mycorrhizal effector PaMiSSP10b alters polyamine biosynthesis in Eucalyptus root cells and promotes root colonization [J]. New Phytol, 2020, 228(2): 712-727.
36	赵龙飞, 徐亚军, 邵璇, 等. 两株内生芽孢杆菌对盐胁迫下大豆幼苗超氧化物歧化酶和过氧化物酶活性影响 [J]. 微生物学通报, 2022, 49(5): 1664-1677.
	Zhao LF, Xu YJ, Shao X, et al. Two endophytic Bacillus strains from soybean nodules affect superoxide dismutase and peroxidase activities in soybean seedlings under salt stress [J]. Microbiol China, 2022, 49(5): 1664-1677.

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