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香草兰(Vanilla planifolia Andrews)属多年生藤本热带经济作物, 享有“香料之王”的誉称,是制作各种高端食品和化妆品等的配香原料。香草兰用途广泛,附加值高,除制作香料外,还具健胃、补肾、软化血管、促进脑功能改善等药用价值,在国际市场上供不应求[1]。香草兰经连续多年种植后,其根际土壤中病原真菌尖孢镰刀菌相对丰度增加;其根际土壤中与枯萎病病情指数呈显著负相关的根际促生菌(PGPR)如芽孢杆菌、木霉、慢生根瘤菌等相对丰度显著降低[2-3]。香草兰经连续种植后,香草兰植株生长受抑,土传枯萎病高发,出现连作生物障碍[2-3]。根系分泌物能够影响植物根际微生物群落组成[4]。植株根系分泌的酚酸类物质是引起根系自毒作用的主要物质,其中酚酸类物质的可溶解部分和可逆吸附的组分是与自毒作用直接相关的[5-6]。在连续种植的花生[7]、甜瓜[8]、烟草[9]、黄瓜[10]的根系分泌物中均检测到酚酸类物质,如苯甲酸、水杨酸、对羟基苯甲酸等。酚酸类物质对种子萌发、植株生长具有显著的抑制作用[11]。西瓜根系分泌的酚酸类物质香草酸、水杨酸和对羟基苯甲酸可促进尖孢镰刀菌孢子萌发和菌丝生长[12]。花生根系分泌物中的苯甲酸被单独添加到根际土壤中时增加了花生根际细菌和真菌群落的丰度,降低了真菌与细菌的比例值,增加了病原真菌尖孢镰刀菌的丰度,使花生的根茎腐病的发病率升高[13]。利用有益微生物降解根系分泌的自毒物质,可降低或消除根系化感物质对土壤及植株的不利影响[14]。刘云露[15]从黄连种植园土壤中分离到能够降解阿魏酸的青霉菌(Penicillium daleae),该菌株培养72 h后对阿魏酸的降解率达87.5%。在花生根际土壤中筛选到1株能够降解多种酚酸的恶臭假单胞菌( Pseudomonas putida),经摇瓶培养5 d时,对酚酸类自毒物质如对羟基苯甲酸、肉桂酸、丁香酸、苯甲酸、阿魏酸等的降解率分别为99.85%、17.44%、90.04%、98. 69%、38.89%[16]。孙秀等[17]在黄瓜根际土壤中也筛选到了能够降解肉桂酸的高效菌株,其中,恶臭假单胞菌(Pseudomonas putida)在12 h内对肉桂酸的降解率达到了99.35%;黑曲霉(Aspergillus niger)在72 h内对肉桂酸的降解率达到了99.62%。目前,国内外对关于自毒物质降解菌的研究主要集中在大宗经济作物上,而较少关注热带经济作物,尤其在多年生热带香料饮料作物上鲜有研究报道。
本课题组前期研究表明,苯甲酸、水杨酸和对羟基苯甲酸是香草兰植株根系分泌的主要自毒物质。本研究从香草兰连作园中筛选到了可降解这3种自毒物质的微生物菌株,通过室内模拟实验研究其降解能力及其对病原菌尖孢镰刀菌的抑制作用,并对其进行了16S rRNA及ITS分子鉴定,为在生产上利用有益微生物缓解香草兰连作生物障碍提供理论基础和菌种资源储备。
Screening, identification and antimicrobial activity of microbial strains degrading autotoxic phenolic acids in the rhizosphere of vanilla
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摘要: 为了探讨利用有益微生物降解根系自毒物质、缓解香草兰连作生物障碍,并储备有益菌种资源,采用传统分离培养方法筛选香草兰根际土壤酚酸类自毒物质降解菌株,并测定其在液体培养基和连作土壤中对酚酸类物质的降解效果及其对病原菌的平板抑制能力。结果表明:从香草兰连作种植园根际土壤筛选到6株可降解酚酸类自毒物质的菌株,分别为真菌BM-5、FD-21、BD-8和细菌ZD-4、ZH-19、ZH-20。菌株FD-21在摇瓶培养72 h时对苯甲酸、对羟基苯甲酸和水杨酸的降解率分别为78.87%、89.5%和93.62%;但在香草兰连作土壤中接种该菌株恒温培养7 d后降解率分别为43.5%、34.2%和67.28%。平板抑菌效果试验表明,该6株菌对香草兰土传枯萎病致病菌尖孢镰刀菌(Fusarium oxysporum f. sp. vanillae )具有一定拮抗作用,其中菌株ZH-19、ZH-20对尖孢镰刀菌的抑制率最高,分别达82.87%和82.94%;FD-21对尖孢镰刀菌的抑制率为45.49%。16S rRNA 和ITS序列比对结果表明,ZD-4为枯草芽孢杆菌(Bacillus subtilis)、ZH-19为炭疽芽孢杆菌(Bacillus anthracis)、ZH-20为解淀粉芽孢杆菌(Bacillus amyloliquefaciens)、BM-5为黄丝曲霉(Talaromyces sp.)、FD-21为青霉(Penicillium sp.)、BD-8为桔青霉(Penicillium citrinum)。说明筛选到的微生物菌株可降解香草兰根系酚酸类自毒物质且对尖孢镰刀菌具有拮抗作用。Abstract: In order to explore the use of beneficial microorganisms to degrade root autotoxic substances, alleviate the biological obstacles of continuous cropping of vanilla (Vanilla planifolia Andr.), and reserve beneficial microbial resources, microbial strains degrading phenolic acids which are autotoxic substances in the rhizosphere of vanilla plantations were screened by using the traditional isolation and culture method. Degrading activity of the isolated microbial strains against the phenolic acids in culture medium and continuous cropping soil and their plate inhibition ability against pathogenic agents were determined, and 6 microbial strains were isolated from the rhizosphere of vanilla plantations with continuous cropping, which were labelled fungi BM-5, FD-21 and BD-8, and bacteria ZD-4, ZH-19 and ZH-20. The fungal strain FD-21 when cultured in shake flasks for 72 h, had degradation rates of 78.87%, 89.5% and 93.62% for p-benzoic acid, p-hydroxybenzoic acid and salicylic acid, respectively, but it had degradation rates of 43.5%, 34.2% and 67.28%, respectively, when thermostatically cultured for 7 days after its inoculation of the continuous cropping soil. The plate bacteriostatic effect test showed that the 6 strains had a certain antagonistic effect on Fusarium oxysporum f. sp. vanillae, a pathogen of vanilla soil-borne Fusarium wilt disease. The bacterial strains ZH-19 and ZH-20 had the highest inhibition rates against F. oxysporum f. sp. vanillae, which were 82.87% and 82.94%, respectively, and the fungal strain FD-21 had an inhibition rate of 45.49% against F. oxysporum f. sp. vanillae. 16S rRNA and ITS sequence alignment showed that the bacterial strain ZD-4 was identified as Bacillus subtilis, the bacterial strain ZH-19 as Bacillus anthracis, the bacterial strain ZH-20 as Bacillus amyloliquefaciens, the fungal strain BM-5 as Talaromyces sp., the fungal strain FD-21 as Penicillium sp., the fungal strain BD-8 as Penicillium citrinum. This indicates that the screened microbial strains can degrade autotoxic phenolic acids secreted by the vanilla roots and have an antagonistic effect on F. oxysporum f. sp. vanillae.
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Key words:
- vanilla /
- autotoxic substance /
- phenolic acid degrading microbe /
- screening /
- identification
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