耐盐碱土曲霉SYAT-1的分离鉴定及抑制植物病原真菌特性研究

doi:10.13560/j.cnki.biotech.bull.1985.2022-0582

摘要/Abstract

摘要：

盐碱地微生物长期在盐碱环境中共存与进化，具有独特适应性和生物学特性，成为微生物改良盐碱地的首选。从吉林省松原市典型苏打盐碱地土壤中分离到1株耐盐碱真菌菌株SYAT-1，通过形态特征观察和ITS-rDNA序列分析对该菌株进行鉴定，并对该菌株不同pH生长特性、耐盐性、拮抗植物病原真菌活性和潜在致病性进行研究。结果表明，该菌株鉴定为土曲霉（Aspergillus terreus）。菌株SYAT-1在碱性（pH 7.0-12.0）培养基条件下均可生长，其生长最适pH范围为7.0-9.0。在0-17.5 mg/mL NaCl 和0-4 mg/mL NaHCO₃的PDA培养基上均能生长。拮抗植物病原真菌测定结果显示，菌株SYAT-1对供试植物病原真菌均有一定的抑制效果。潜在致病性测试结果表明，菌株SYAT-1对供试植物无潜在的致病性。土曲霉菌株SYAT-1属于耐高盐碱特性的真菌，并具有抑制植物病原真菌的特性，该结果为利用微生物改良盐碱土和植物病害生物防治提供新的菌株资源和理论支撑。

关键词: 耐盐碱, 土曲霉, 分离鉴定, 病原真菌, 抑制

Abstract:

Microorganisms co-exist and evolve in saline environment for a long time and have unique adaptability and biological properties, which make them the first choice in the microbial improvement of saline land. A saline-alkali tolerant fungal strain SYAT-1 was isolated from a typical soda saline soil in Songyuan city, Jilin province. It was identified by morphological characteristics observation and ITS-rDNA sequence analysis. The growth characteristics at different pH, salt tolerance, antagonistic activity against phytopathogenic fungi and potential pathogenicity of the strain were investigated. The results showed that it was identified as Aspergillus terreus and grew under alkaline（pH=7.0-12.0）medium conditions, with an optimal pH range of 7.0-9.0. It grew well on both 0-17.5 mg/mL NaCl PDA medium and 0-4mg/mL NaHCO₃ PDA medium. Antagonistic phytopathogenic fungi assay results revealed that strain SYAT-1 presented some inhibitory effect on the test phytopathogenic fungi. Potential pathogenicity test results demonstrated that strain SYAT-1 had no potential pathogenicity to the test plants. In sum, A. terreus strain SYAT-1 belongs to a fungus with high salt tolerance characteristics and suppressing phytopathogenic fungi. This study provides new strain resources and theoretical support for the use of microorganisms to improve saline soils and biological control of plant diseases.

Key words: saline-alkali tolerance, Aspergillus terreus, isolation and identification, pathogenic fungus, inhibition

王凤婷, 王岩, 孙颖, 崔文婧, 乔凯彬, 潘洪玉, 刘金亮. 耐盐碱土曲霉SYAT-1的分离鉴定及抑制植物病原真菌特性研究[J]. 生物技术通报, 2023, 39(2): 203-210.

WANG Feng-ting, WANG Yan, SUN Ying, CUI Wen-jing, QIAO Kai-bin, PAN Hong-yu, LIU Jin-liang. Isolation and Identification of Saline-alkali Tolerant Aspergillus terreus SYAT-1 and Its Activities Against Plant Pathogenic Fungi[J]. Biotechnology Bulletin, 2023, 39(2): 203-210.

图/表 9

表1 采集与分离的真菌菌株

Table 1 Collected and isolated fungal strains

土壤编号 Soil No.	采样时间 Collection time	菌株编号 Strain code	土壤编号 Soil No.	采样时间 Collection time	菌株编号 Strain code
SY-1	2020.8.10	SYAT-1	SY-8	2019.9.16	SYAT-4
SY-1	2020.8.10	SYA-2	SY-9	2019.9.16	SYA-1
SY-2	2020.8.10	SYAT-3	SY-9	2019.9.16	SYAO-3
SY-3	2020.8.10	SYTA-1	SY-10	2019.9.16	SYAT-2
SY-3	2020.8.10	SYA-5			SYA-4
SY-4	2020.8.10	SYTH-1			SYAN-1
SY-4	2020.8.10	SYA-3	SY-11	2019.9.16	SYTH-2
SY-5	2020.8.10	SYAO-1	SY-11	2019.9.16	SYAN-2
SY-6	2020.8.10	SYTA-3	SY-12	2019.9.16	SYTA-2
SY-7	2020.8.10	SYA-6	SY-12	2019.9.16	SYA-6
SY-7	2020.8.10	SYAO-2

图1 菌株SYAT-1形态特征 A：菌落形态；B：分生孢子梗；C：分生孢子

Fig. 1 Morphological characteristics of strain SYAT-1 A：Colony morphology. B：Conidial peduncle. C：Conidiophore

图2 基于ITS-rDNA 核苷酸序列构建的系统进化树 Bootstrap值（%）大于50的显示。系统树上各个菌株表示为：登录号-菌株名称-菌株号

Fig. 2 Phylogenetic tree constructed based on ITS-rDNA nucleotide sequences Bootstrap values（%）> 50 were displayed. The individual strain in the system tree was represented as：GenBank number-Strain name-Strain number

图3 菌株SYAT-1在不同pH下生长不同小写字母表示在0.05水平差异显著。下同

Fig. 3 Growth of strain SYAT-1 at different pH Different lower letters indicate significant differences at 0.05 level. The same below

图4 菌株SYAT-1 28℃不同浓度NaCl浓度生长情况

Fig. 4 Growth of strain SYAT-1 at 28℃ with different concentrations of NaCl

图5 菌株SYAT-1 28℃不同浓度NaHCO3下生长情况

Fig. 5 Growth of strain SYAT-1 at 28℃ with different concentrations of NaHCO3

图6 菌株SYAT-1对不同病原真菌的拮抗效果（a）：核盘菌；（b）：禾谷镰孢；（c）：灰葡萄孢；（d）：玉米小班病菌；（e）：玉米大斑病菌；（f）稻瘟病菌

Fig. 6 Antagonistic effect of strain SYAT-1 against diffe-rent pathogenic fungi （a）：Sclerotinia sclerotiorum；（b）：Fusarium graminearum；（c）：Botrytis cinerea；（d）：Cochliobolus heterostrophus；（e）：Setosphaeria turcica；（f）：Magnaporthe oryzae

表2 菌株SYAT-1对6种植物病原真菌的抑菌率

Table2 Inhibition rates of strain SYAT-1 against six plant pathogenic fungi

植物病原真菌Plant pathogenic fungus	抑菌率Inhibition rate/%
Sclerotinia sclerotiorum	34.12±0.81d
Fusarium graminearum	35.29±0.69d
Botrytis cinerea	42.35±0.65c
Cochliobolus heterostrophus	47.89±0.33b
Setosphaeria turcica	50.59±0.74a
Magnaporthe oryzae	21.57±0.73e

图7 菌株SYAT-1潜在致病性测试 A：核盘菌接种烟草、番茄和大豆叶片；B：土曲霉SYAT-1接种烟草、番茄和大豆叶片；C1，C2：玉米大斑病菌孢子悬浮液接种玉米叶片；C3，C4：接种土曲霉SYAT-1孢子悬浮液；C5：接种20% Tween-20作为对照

Fig. 7 Potential pathogenicity test of the strain SYAT-1 A：Tobacco，tomato and soybean leaves inoculated with S. sclerotiorum. B：Tobacco，tomato and soybean leaves inoculated with A. terreus SYAT-1. C1，C2：Maize leaves inoculated with spore suspension of S. turcica. C3，C4：Maize leaves inoculated with spore suspension of A. terreus SYAT-1. C5：Maize leaves inoculation with 20% Tween-20 as control

参考文献 25

[1]	王佳丽, 黄贤金, 钟太洋, 等. 盐碱地可持续利用研究综述[J]. 地理学报, 2011, 66(5):673-684.
	Wang JL, Huang XJ, Zhong TY, et al. Review on sustainable utilization of salt-affected land[J]. Acta Geogr Sin, 2011, 66(5):673-684. doi: 10.11821/xb201105010
[2]	Li JG, Pu LJ, et al. Soil salinization research in China:advances and prospects[J]. J Geogr Sci, 2014, 24(5):943-960. doi: 10.1007/s11442-014-1130-2 URL
[3]	Liu SL, Maimaitiaili B, Joergensen RG, et al. Response of soil microorganisms after converting a saline desert to arable land in central Asia[J]. Appl Soil Ecol, 2016, 98:1-7. doi: 10.1016/j.apsoil.2015.08.024 URL
[4]	杨劲松, 姚荣江. 我国盐碱地的治理与农业高效利用[J]. 中国科学院院刊, 2015, (201):162-170
	Yang JS, Yao RJ. Management and efficient agricultural utilization of salt-affected soil in China[J]. Bulletin of the Chinese Academy of Sciences, 2015, (201):162-170.
[5]	任培根, 周培瑾. 中度嗜盐菌的研究进展[J]. 微生物学报, 2003, 43(3):427-431.
	Ren PG, Zhou PJ. Reseach progress of moderately halophilic eubacteria[J]. Acta Microbiol Sin, 2003, 43(3):427-431.
[6]	Gunde-Cimermana N, Zalarb P, De HS, et al. Hypersaline waters in salterns - natural ecological niches for halophilic black yeasts[J]. FEMS Microbiol Ecol, 2000, 32(3):235-240. pmid: 10858582
[7]	Kis-Papo T, Oren A, et al. Survival of filamentous fungi in hypersaline Dead Sea water[J]. Microb Ecol, 2003, 45(2):183-190. pmid: 12545316
[8]	Wei Y, Zhang SH. Abiostress resistance and cellulose degradation abilities of haloalkaliphilic fungi:applications for saline-alkaline remediation[J]. Extremophiles, 2018, 22(2):155-164. doi: 10.1007/s00792-017-0986-3 pmid: 29290045
[9]	黄瑞环, 韩小斌, 刘京, 等. 海洋曲霉和海洋木霉抗植物病原菌活性次级代谢产物研究进展[J]. 江苏农业学报, 2020, 36(5):1332-1341.
	Huang RH, Han XB, Liu J, et al. Research progress on secondary metabolites with anti-phytopathogenic activities of marine-derived Aspergillus sp and Trichoderma sp[J]. Jiangsu J Agric Sci, 2020, 36(5):1332-1341.
[10]	李瑾, 彭可为, 潘求一, 等. 解淀粉芽胞杆菌HR-2的分离鉴定及对水稻稻瘟病菌的拮抗效果[J]. 生物技术通报, 2021, 37(3):27-34. doi: 10.13560/j.cnki.biotech.bull.1985.2020-0970
	Li J, Peng KW, Pan QY, et al. Isolation and identification of Bacillus amyloliquefaciens HR-2 and biological control of rice blast[J]. Biotechnol Bull, 2021, 37(3):27-34.
[11]	祖雪, 周瑚, 朱华珺, 等. 枯草芽孢杆菌K-268的分离鉴定及对水稻稻瘟病的防病效果[J]. 生物技术通报, 2022, 38(6): 136-146.
	Zu X, Zhou H, Zhu HJ, et al. Isolation and identification of Bacillus subtilis K-268 and its biological control effect on rice blast[J]. Biotechnol Bull, 2022, 38(6): 136-146.
[12]	马小翔, 刘亚月, 聂影影, 等. 基于质谱的分子网络分析化学调控对土曲霉C23-3次生代谢产物及生物活性的影响[J]. 生物技术通报, 2021, 37(8):95-110. doi: 10.13560/j.cnki.biotech.bull.1985.2020-1398
	Ma XX, Liu YY, Nie YY, et al. LC-MS/MS based molecular network analysis of the effects of chemical regulation on the secondary metabolites and biological activities of a fungal strain Aspergillus terreus C23-3[J]. Biotechnol Bull, 2021, 37(8):95-110.
[13]	White TJ, Bruns T, Lee S, et al. Amplification and direct sequencing of fungal ribosomal RNA genes for phylogenetics[M]// PCR Protocols Amsterdam: Elsevier, 1990:315-322.
[14]	刘冰, 周红霞, 赵美茜, 等. 玉米茎腐病生防菌株的筛选及抑菌作用研究[J]. 玉米科学, 2021, 29(6):164-168.
	Liu B, Zhou HX, Zhao MX, et al. Screening of biocontrol strains against corn stalk rot and antifungal mechanisms[J]. J Maize Sci, 2021, 29(6):164-168.
[15]	迪力热巴·阿不都肉苏力, 穆耶赛尔·奥斯曼, 祖力胡玛尔·肉孜, 等. 盐碱土壤微生物多样性与生物改良研究进展[J]. 生物技术通报, 2021, 37(10):225-233. doi: 10.13560/j.cnki.biotech.bull.1985.2020-1344
	Dilireba A, Muyesaier A, Zulihumaer R, et al. Advances on microbial diversity and biological improvement of saline-alkali soil[J]. Biotechnol Bull, 2021, 37(10):225-233. doi: 10.13560/j.cnki.biotech.bull.1985.2020-1344
[16]	刘晓丹, 刘金亮, 魏毅, 等. 一株极端耐盐曲霉的分离、鉴定及生物学特性分析[J]. 吉林大学学报:理学版, 2011, 49(3):548-553.
	Liu XD, Liu JL, Wei Y, et al. Isolation, identification and biologic characteristics of an extreme halotolerant Aspergillus sp[J]. J Jilin Univ Sci Ed, 2011, 49(3):548-553.
[17]	郑晓梅, 丁旭扬, 黄婷, 等. 一株产酸黑曲霉的分离鉴定及其在盐碱土改良中的应用[J]. 山东农业科学, 2019, 51(4):69-73, 78.
	Zheng XM, Ding XY, Huang T, et al. Isolation and identification of an acid-producing Aspergillus niger and its application in improvement of saline-alkali soil[J]. Shandong Agric Sci, 2019, 51(4):69-73, 78.
[18]	李学平, 谢文军, 范延辉. 盐碱地塔宾曲霉菌的解磷能力及其对小麦生长的影响[J]. 水土保持通报, 2017, 37(1):93-96.
	Li XP, Xie WJ, Fan YH. Phosphate-solubilizing ability of Aspergillus tubingensis and its effects on growth of wheat in seedling stage[J]. Bull Soil Water Conserv, 2017, 37(1):93-96.
[19]	王丹, 芶剑渝, 韩小斌, 等. 海洋真菌次级代谢产物在植物保护中的研究与应用[J]. 中国生物防治学报, 2019, 35(1):146-158.
	Wang D, Gou JY, Han XB, et al. Research and application of secondary metabolites from marine-derived fungus in plant protection[J]. Chin J Biol Control, 2019, 35(1):146-158.
[20]	黄庶识, 等. 3株抗水稻和荔枝病原菌的海洋真菌的分离鉴定[J]. 基因组学与应用生物学, 2010, 29(4):665-672.
	Huang SS, et al. Isolation and identification of 3 strains of marine fungi with antagonistic ability against Litchi and rice pathogenic fungi[J]. Genom Appl Biol, 2010, 29(4):665-672.
[21]	孙好芬. 两株热带马尾藻内生真菌次生代谢产物研究[D]. 青岛: 中国科学院研究生院(海洋研究所), 2012.
	Sun HF. Optimization of culture conditions and secondary metabolites of two Sargassum sp derived endophytic fungus[D]. Qingdao: Institute of Oceanology, Chinese Academy of Sciences, 2012.
[22]	杨遂群. 五株海藻及红树林来源真菌次级代谢产物的分子多样性挖掘与生物活性研究[D]. 北京: 中国科学院大学, 2018.
	Yang SQ. Chemical diversity and biological evaluation of secondary metabolites of five fungul strains derived from marine algae and mangrove[D]. Beijing: University of Chinese Academy of Sciences, 2018.
[23]	李祝, 肖洋, 邱红波. 黑曲霉绿色防控植物病害菌剂创制及应用[Z]. 贵阳: 贵州大学, 2019.
	Li Z, Xiao Y, Q HB. The creation and application of Aspergillus niger green control of plant disease fungicide[Z]. Guiyang: Guizhou University, 2019.
[24]	刘警鞠, 张雨森, 陈娟, 等. 曲霉属的现代分类命名研究进展[J]. 生物技术通报, 2022, 38(7): 109-118.
	Liu JL, Zhang YS, Chen J, et al. Research progress in modern taxonomy and nomenclature of Aspergillus.[J]. Biotechnol Bul, 2022, 38(7): 109-118.
[25]	沙娜瓦尔·色买提, 玉山江·买买提, 郭庆元, 等. 枣果霉烂病病原鉴定(二)——引起新疆枣果霉烂病的几种青霉菌的分离鉴定[J]. 新疆农业科学, 2016, 53(4):698-705.
	Shanawaer S, Yushanjiang M, Guo QY, et al. Identification of the pathogen causing jujube fruit mildew(part II)—isolation and identification of Penicillium fungus causing jujube fruit mildew[J]. Xinjiang Agric Sci, 2016, 53(4):698-705.