首页 > 分享 > 一株土壤来源的贝莱斯芽孢杆菌抑制黄曲霉生长机理研究

一株土壤来源的贝莱斯芽孢杆菌抑制黄曲霉生长机理研究

花匠小妙招
2024-11-14 23:18

摘要: 为了更加安全有效地防治黄曲霉及其毒素污染,从花生田地土壤中分离获得了一株对黄曲霉生长具有良好抑制作用的菌株,通过平板对峙试验,验证其拮抗黄曲霉的效果;通过16S rDNA序列分析、构建进化树以及生理生化指标明确菌株种属信息;通过发酵优化、孢子萌发试验,发现该菌株在葡萄糖培养基、蔗糖培养基、牛肉膏培养基中的发酵液均能抑制黄曲霉孢子萌发,其中在葡萄糖培养基中的发酵液抑制效果最强,并考察了其对黄曲霉菌丝生长及孢子产生的抑制作用;采用PI和DAPI染色探讨了发酵液抑制黄曲霉菌丝生长的机理。结果表明:分离获得的菌株在平板对峙试验中能够显著抑制黄曲霉菌落生长,该菌株为革兰氏阳性菌株贝莱斯芽孢杆菌;在葡萄糖培养基中发酵48 h获得的发酵液对黄曲霉孢子萌发抑制率最佳,达到79.35%;在固体PDA培养基上对黄曲霉菌落生长抑制率为29%,对孢子生成的抑制率为54%;染色结果显示,发酵液中的抑菌物质能够通过破坏黄曲霉细胞膜的通透性以及损伤DNA的方式抑制菌丝生长。该菌株及其发酵液对黄曲霉具有较好的防治作用,具有进一步开发为农产品防霉剂的潜力。

Abstract: In order to control Aspergillus flavus growth and aflatoxin contamination more safely and effectively, a strain with excellent inhibitory effect on the growth of A. flavus was isolated from the soil of peanut field. The antagonistic effect against A. flavus was confirmed by plate confrontation examination; 16S rDNA sequence alignment analysis, physiological and biochemical characterization were employed for strain identification. Furthermore, the optimal medium for producing antibacterial substance was evaluated by spore germination investigation, and the inhibition effect of fermentation broth on mycelial growth and spore production of A. flavus was evaluated. In addition, the inhibitory mechanism was elucidated through PI and DAPI staining. The results showed that the colony growth of A. flavus was inhibited by strain 41 in a noncontact mode, indicating that strain 41 could antagonize A. flavus via producing some antifungal compounds. The physiological and biochemical analysis showed that strain 41 was a gram-positive bacterium and could utilize glucose, maltose, fructose, sucrose, and xylose, as well as hydrolyze starch. In salt-tolerance experiments, strain 41 grew normally in 2% NaCl, 5% NaCl, and 7% NaCl, but not in media containing 10% NaCl. Furthermore, strain 41 could inhibit spore germination of A. flavus in glucose medium, sucrose medium and beef extract medium, among which glucose medium was the most effective, and the inhibitory effects of the fermentation broth on the mycelial growth and spore production of A. flavus were investigated under this condition. The inhibition rate of A. flavus spore germination was 79.35% by fermentation broth obtained in glucose medium of the isolated strain for 48 hours. On solid PDA medium, the inhibition rate of A. flavus colony growth and spore production was 29% and 54% respectively. The results of PI and DAPI staining on the mycelia treated with the fermentation broth showed that the antibacterial substances in the fermentation broth could inhibit the growth of A. flavus mycelia by destroying the permeability of cell membrane and damaging DNA respectively. This study demonstrated that the strain and its fermentation broth had an excellent inhibitory effect on A. flavus and provided a theoretical basis for development of the antifungal substance as a mildew inhibitor in agricultural field.

[1] 李金芝.黄曲霉拮抗菌株的筛选、鉴定及其活性物质的初步研究[D].青岛:青岛科技大学, 2013. [2] 王后苗.花生抗黄曲霉菌产毒机制的研究[D].北京:中国农业科学院, 2016. [3] 王后苗,廖伯寿,雷永,等.黄曲霉菌主要真菌毒素次级代谢与调控的研究进展[J].微生物学通报, 2014, 41(7):1425-1438. [4]

JALLOW A, XIE H L, TANG X Q, et al. Worldwide aflatoxin contamination of agricultural products and foods:from occurrence to control[J]. Comprehensive reviews in food science and food safety, 2021, 20(3):2332-2381.

[5] 刘东璞,卢凤美,姚海涛,等.黄曲霉毒素B1诱导大鼠肝癌模型的建立[J].黑龙江医药科学, 2012, 35(6):47-48. [6] 吴佳遥,李磊,游海平,等.黄曲霉毒素的危害及植物精油的抑制作用进展[J].天津农业科学, 2019, 25(7):61-64. [7] 屈璐璐.芽孢杆菌所产挥发性物质对黄曲霉的抑制作用研究[D].长沙:中南林业科技大学, 2018. [8] 王昌禄,刘彤,李王强,等.微生物对黄曲霉毒素的抑制机制研究进展[J].食品科学技术学报, 2021, 39(1):27-36. [9] 周启升,马峰,鞠瑞成,等.饲料防霉拮抗菌的筛选[J].中国畜牧业, 2021(17):52-53. [10] 曹利,车程川,刘金锋,等.拮抗黄曲霉海洋放线菌的筛选及发酵条件优化[J].中国酿造, 2019, 38(3):104-109. [11] 李天溪,党萌,龙淼. 1株黄曲霉颉颃菌的鉴定及其抑菌活性成分分析[J].中国畜牧兽医, 2022, 49(5):1828-1839. [12] 袁远,张佩,何鹏飞,等.黄曲霉毒素B1降解菌的筛选及在玉米贮藏中的应用[J].微生物学报, 2019, 59(2):395-404. [13] 姚彦坡,丁丹,张友青,等.玉米黄曲霉毒素污染生防菌筛选及菌株B42-3抗菌活性研究[J].中国粮油学报, 2018, 33(3):84-88. [14]

CAO Y, PI H L, CHANDRANGSU P, et al. Antagonism of two plant-growth promoting Bacillus velezensis isolates against Ralstonia solanacearum and Fusarium oxysporum[J]. Scientific reports, 2018, 8:4360.

[15] 张彩文,程坤,张欣,等.贝莱斯芽胞杆菌(Bacillus velezensis)分类学及功能研究进展[J].食品与发酵工业,2019,45(17):258-265. [16] 闫浩浩.贝莱斯芽胞杆菌SDTB038的分离鉴定及其生防作用的研究[D].泰安:山东农业大学, 2021. [17] 吕昂.小麦PINA蛋白体外表达及其抗黄曲霉作用研究[D].郑州:河南工业大学, 2019. [18] 汪雪静,卜春亚,靳永胜,等.草莓根腐病菌拮抗细菌的分离与鉴定[J].园艺学报, 2011, 38(9):1657-1666. [19] 隋松森,王松江,郭传庄,等.微生物发酵法产赤藓糖醇的研究进展[J].中国食品添加剂, 2021, 32(6):125-131. [20] 张敏,郑媛,赵玉洋,等.假单胞菌菌株ZL8发酵培养基及发酵条件的优化[J].中国实验方剂学杂志, 2022, 28(22):174-181. [21] 薛一达,丛丽娜,李若凡.海洋链霉菌HS-B31发酵条件优化及活性物质分离纯化[J].大连工业大学学报, 2020, 39(4):245-249. [22]

HUSZCZA E, BURCZYK B. Surfactin isoforms from Bacillus coagulans[J]. Zeitschrift fur naturforschung C, 2006, 61(9/10):727-733.

[23] 靳佳琦,闻建平.合成生物学指导下芽孢杆菌合成环脂肽的研究进展[J].中国生物工程杂志, 2022, 42(6):86-101. [24]

BAIS H P, FALL R, VIVANCO J M. Biocontrol of Bacillus subtilis against infection of Arabidopsis roots by Pseudomonas syringae is facilitated by biofilm formation and surfactin production[J]. Plant physiology, 2004, 134(1):307-319.

[25]

MUKHERJEE A K, DAS K.. Correlation between diverse cyclic lipopeptides production and regulation of growth and substrate utilization by Bacillus subtilis strains in a particular habitat[J]. FEMS microbiology ecology, 2005, 54(3):479-489.

[26]

ONGENA M, JOURDAN E, ADAM A, et al. Surfactin and fengycin lipopeptides of Bacillus subtilis as elicitors of induced systemic resistance in plants[J]. Environmental microbiology, 2007, 9(4):1084-1090.

[27]

SANTOS V S V, SILVEIRA E, PEREIRA B B. Toxicity and applications of surfactin for health and environmental biotechnology[J]. Journal of toxicology and environmental health, part B, 2018, 21(6/7/8):382-399.

[28] 李苏冉,李雪,冯佳霖,等.生防菌解淀粉芽孢杆菌SQ-2全基因组测序及生物信息学分析[J/OL].[2022-11-11].微生物学通报, https://kns.cnki.net/KCMS/detail/detail.aspx?filename=WSWT20221109004&dbname=CJFD&dbcode=CJFQ. [29]

DELEU M, PAQUOT M, NYLANDER T. Effect of fengycin, a lipopeptide produced by Bacillus subtilis, on model biomembranes[J]. Biophysical journal, 2008, 94(7):2667-2679.

[30] 陆景倩,郎剑锋,杨秋侠,等.解淀粉芽孢杆菌对植物土传病害的作用机制[J].湖北农业科学,2021,60(12):5-10.