短小芽孢杆菌AR03挥发性有机物的抑菌活性及其组分分析

doi:10.3864/j.issn.0578-1752.2018.10.010

摘要/Abstract

摘要：

目的对分离自烟草根际土壤中的一株短小芽孢杆菌（Bacillus pumilus）AR03菌株产生的挥发性有机物进行抑菌活性测定和组分分析。方法采用培养皿对扣熏蒸法和凹玻片孢子萌发法测定挥发性有机物对烟草黑胫病菌（Phytophthora parasitica var. nicotianae）和赤星病菌（Alternaria alternata）菌落、菌丝生长以及孢子萌发的影响,采用离体叶片接种法测定挥发性有机物对烟草黑胫病和赤星病的防治效果;采用顶空加热法收集抑菌挥发性有机物,对其进行气相色谱-质谱（GC-MS)检测,并结合保留指数（retention index,RI）和内标（internal standard,IS）1-十五烯对其组分进行定性和定量分析。结果短小芽孢杆菌AR03菌株分泌的挥发性有机物对供试的两种病原真菌均具有一定的抑制作用,表现为经处理后的烟草黑胫病菌菌丝生长稀疏缓慢变粗、分支增多且扭曲、断裂;赤星病菌菌丝生长畸形,其上不产生分生孢子梗,内含物大部分聚集成团导致菌丝干瘪、缢缩。对峙培养2、4、6和8 d时,挥发物对黑胫病菌和赤星病菌菌丝生长的抑制率分别为56.21%和59.23%、64.75%和59.86%、66.13%和61.10%、67.04%和70.00%;对黑胫病菌游动孢子和赤星病菌分生孢子的抑制作用表现为延迟萌发且萌发后的芽管生长缓慢;黑胫病菌产生孢子囊数明显减少,赤星病菌分生孢子各分隔处的细胞畸形肿胀成泡状。离体叶片抗病测定结果显示,挥发性有机物抑制黑胫病菌和赤星病菌病斑在叶片的扩展,对峙熏蒸40 h时,对照叶片黑胫病的发病率为92.50%,圆形坏死病斑直径是25.10 mm,经挥发物处理后叶片发病率为70.83%,病斑直径为9.45 mm,对黑胫病斑扩散的抑制率为62.35%;对峙熏蒸80 h时,对照叶片赤星病的发病率为88.33%,病斑扩散为近圆形的褐色坏死斑,平均直径为8.32 mm,经挥发物处理后赤星病的发病率为60.80%,病斑扩展较慢,平均直径为2.85 mm,挥发物对赤星病斑扩散的抑制率为65.75%。SPME GC-MS分析表明,AR03菌株胞外分泌7种组分挥发性物质,均为C₁₅H₂₄结构的倍半萜烯类化合物,分别是二氢姜黄烯、(E)-β-金合欢烯、γ-姜黄烯、α-姜烯、π-红没药烯、β-倍半萜水芹烯和γ-E-红没药烯;AR03培养1 d后,其中β-倍半萜水芹烯相对含量最高,为80.64%,其次为(E)-β-金合欢烯和α-姜烯,相对含量分别为7.20%和6.67%;随着AR03培养时间的延长,各组分种类相同,但相对含量差异较大,除二氢姜黄烯含量保持基本不变外,其他组分含量呈递减趋势,培养至第6天时,各组分含量下降超过50%。结论短小芽孢杆菌AR03菌株能够产生具有较强抑菌活性的挥发性有机物,有潜力作为开发抗真菌代谢物和新药物的重要微生物资源。

关键词: 短小芽孢杆菌AR03, 挥发性有机物, 烟草黑胫病菌, 烟草赤星病菌, 顶空-固相微萃取-气质联用

Abstract:

【Objective】 The objective of this study is to determine the antimicrobial activity of volatile organic compounds (VOCs), produced from tobacco rhizosphere soil Bacillus pumilus AR03 strain, and to analyze its main components.【Method】 The antifungal effect of VOCs on the colony, mycelium growth and spore germination of Phytophthora parasitica var. nicotianae and Alternaria alternata was determined by a double Petri dish assay and cavity slide method. The control effect of VOCs on tobacco black shank and brown spot by leaf inoculation was determined in vitro. VOCs were collected by head-space solid phase microextraction (HS-SPME) and identified by gas chromatography-mass spectrometry (GC-MS). Retention index (RI) and internal standard (IS) 1-pentadecence were used for qualitative and quantitative analysis.【Result】 VOCs released from B. pumilus AR03 strain had certain inhibitory effect on the two target pathogens, which showed that the mycelium of P. parasitica grew slowly and thinned, branches increased and twisted, and broke. The mycelium of A. alternata was deformed and no conidial pedicle was produced on the mycelium. Most of the inclusions gathered together and caused the mycelium to dry and constricted. Growth of exposed fungus colonies was inhibited by VOCs, the inhibition rates of VOCs were 56.21% and 59.23%, 64.75% and 59.86%, 66.13% and 61.10%, 67.04% and 70.00%, respectively, against P. parasitica and A. alternata cultured in sealed plates for 2, 4, 6 and 8 d. When the zoospores of P. parasitica and ascospores of A. alternata exposed to these volatile components for 6 h, the germination was delayed and the growth was slow. The number of sporocyst produced by P. parasitica obviously reduced. Most conidiophores of A. alternata expanded abnormally as cystic structure, indicating the fungicidal nature of the volatiles. Moreover, VOCs could significantly inhibit the disease severities of tobacco black shank and brown spot on leaves tests. Direct fumigation for 40 h and 80 h, black shank disease incidence was 92.50% on control and 70.83% on leaves treated by VOCs, the inhibitory of spot expansion was 62.35%. Brown spot disease incidence was 88.33% on control and 60.80% on leaves treated by VOCs, lesions expanded slowly and inhibitory rate was 65.75%. SPME GC-MS analysis showed that seven components of the volatiles were identified, all of which are sesquiterpenes with C₁₅H₂₄structure. They are dihydrocurcumene (CAS NO. 1461-02-5), (E)-β-famesene (CAS NO. 18794-84-8), γ-curcumene (CAS NO. 451-55-8), α-zingiberene (CAS NO. 495-60-3), π-bisabolene (CAS NO. 495-61-4), β-sesquiphellandrene (CAS NO. 20307-83-9) and γ-E-bisabolene (CAS NO. 53585-13-0). When AR03 was cultured for 1 d, the relative content of β-sesquiphellandrene was the highest (80.64%), followed by (E)-β-famesene and α-zingiberene, the relative content was 7.20% and 6.67%, respectively. With the extension of culturing time, the species of each component were the same, but the relative content was different. Except for dihydrocurcumene, the content of other components showed a decreasing trend, when cultured for 6 d, other ingredients decreased more than 50%, besides dihydrocurcumene keeping relatively stable. 【Conclusion】 VOCs produced by B. pumilus AR03 could develop an additive antifungal effect against fungal pathogens on tobacco. B. pumilus AR03 has potential as an important microbial resource for developing antifungal metabolites and new drugs.

Key words: Bacillus pumilus AR03, volatile organic compounds, Phytophthora parasitica var. nicotianae, Alternaria alternata, SPME GC-MS

王静, 曹建敏, 陈德鑫, 邱军, 王晓强, 冯超, 王文静. 短小芽孢杆菌AR03挥发性有机物的抑菌活性及其组分分析[J]. 中国农业科学, 2018, 51(10): 1908-1919.

Jing WANG, JianMin CAO, DeXin CHEN, Jun QIU, XiaoQiang WANG, Chao FENG, WenJing WANG. Antimicrobial Effect and Components Analysis of Volatile Organic Compounds from Bacillus pumilus AR03[J]. Scientia Agricultura Sinica, 2018, 51(10): 1908-1919.

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处理 Treatment	叶片Leave
	烟草黑胫病Tobacco black shank			烟草赤星病Tobacco brown spot
	病斑直径 Lesion diameter (mm)	发病率 Incidence (%)	抑制率 Inhibition rate (%)	病斑直径 Lesion diameter (mm)	发病率 Incidence (%)	抑制率 Inhibition rate (%)
挥发性有机物VOCs	9.45	70.83	62.35	2.85	60.80	65.75
CK	25.10	92.50	—	8.32	88.33	—

峰编号 Peak number	保留时间Retention time (min)	名称 Name	结构式 Structural formula	CAS编号 CAS number	保留指数（计算） RI (calculation)	保留指数（文献） RI (reference)
1	22.824	二氢姜黄烯Dihydrocurcumene	C₁₅H₂₄	1461-02-5	1449	1448
2	23.061	(E)-β-金合欢烯(E)-β-Famesene	C₁₅H₂₄	18794-84-8	1458	1457
3	23.642	γ-姜黄烯γ-Curcumene	C₁₅H₂₄	451-55-8	1482	1479
4	24.024	α-姜烯α-Zingiberene	C₁₅H₂₄	495-60-3	1497	1500
5	24.353	π-红没药烯π-Bisabolene	C₁₅H₂₄	495-61-4	1511	1509
6	24.720	β-倍半萜水芹烯β-Sesquiphellandrene	C₁₅H₂₄	20307-83-9	1526	1525
7	24.914	γ-E-红没药烯γ-E-Bisabolene	C₁₅H₂₄	53585-13-0	1535	1535
内标IS	23.906	1-十五烯1-Pentadecene	C₁₅H₃₀	13360-61-7	—	—

名称 Name	倍半萜烯代谢物含量 Relative contents of sesquiterpenes (ng)
名称 Name	1 d	3 d	6 d
二氢姜黄烯Dihydrocurcumene	1.07	1.10	1.13
(E)-β-金合欢烯(E)-β-Famesene	10.00	6.99	4.24
γ-姜黄烯γ-Curcumene	2.73	1.88	1.43
α-姜烯α-Zingiberene	9.27	6.32	4.60
π-红没药烯π-Bisabolene	1.74	1.35	1.05
β-倍半萜水芹烯β-Sesquiphellandrene	112.01	82.03	54.66
γ-E-红没药烯γ-E-Bisabolene	2.08	1.27	0.76