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油茶(Camellia oleifera Abel.)属山茶科(Theaceae)山茶属(Cameuia)植物,是我国主要的经济林树种,也是世界四大木本油料植物之一[1]。由于油茶具有适应性强、抗逆性好的特性,十分适宜在相对贫瘠的红壤丘陵地区种植,因此广泛分布于我国湖南、江西、广西等17个省区,在我国已有超过两千年的种植历史。我国现有油茶林种植面超过4.3×106 hm2,油茶籽年产达217万吨,产值超过600亿元。湖南作为油茶的最大产区和茶油的最大消费区,油茶资源约占全国40%[2]。但是,油茶的种植生产过程中往往受到多种病虫害的侵袭,导致油茶的产量和品质大幅下降,其中以油茶炭疽病对油茶林的危害最为严重。
油茶炭疽病(Colletotrichum camellia Massee)病菌能够侵染油茶的花芽、叶芽、果实、枝梢和叶片,通常引起油茶的落花、落果和枝枯现象,严重时甚至能造成整株死亡;在我国各油茶产区均有发生,可导致20%左右的平均落果率,有时可高达40%~50%,是油茶种植过程中的最主要病害之一[3]。尽管化学农药对油茶炭疽病的防治具有一定效果[4-7],但长期滥用化学农药会引起病原菌的抗药性,造成环境污染,同时大幅增加成本[8-9]。因此,开发出对油茶炭疽病防治效果显著,无污染、无公害、可持续的生物防治手段显得尤为重要。
芽孢杆菌属中的解淀粉芽孢杆菌(Bacillus amyloliquefaciens)在自然界中分布广泛,对人、畜无毒无害,具有很高的抗逆能力;研究表明,解淀粉芽孢杆菌具有广谱的抑制真菌和细菌活性的作用,且生长速度快、稳定性强,是一种理想的生防菌资源[10-11]。解淀粉芽孢杆菌的主要生防机制包括有定殖、分泌拮抗物质、诱导系统抗性等,其中分泌拮抗物质如脂肽类抗生素和挥发性物质等,可显著抑制病原微生物的活性,抑制病害的发生[12-13]。因此,本研究分别通过高效液相色谱法(HPLC)和气相色谱-质谱法(GC-MS)对前期研究工作中分离得到的解淀粉芽孢杆菌菌株P-14中的脂肽类抗生素和挥发性物质分别进行分离、鉴定,以期获得高效的抑菌物质用于油茶炭疽病的生物防治。
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通过HPLC分析,从解淀粉芽孢杆菌P-14发酵液中纯化收集到5种主要组分,其分析图谱如图 1所示;对收集的5种组分进行冷冻干燥后,溶于一定量的甲醇中备用。运用牛津杯法分别测定5种组分的抗菌活性,抗菌效果如图 2所示,结果表明5种主要成分中仅有a号峰有抑菌活性。将收集的a号峰进行HPLC-ESI-MS分析,得到分子量分别为1 045.5和1 067.5的物质,其质谱图如图 3所示。对比芽孢杆菌脂肽物质的分子量数据可知:组分a中主要是含有C15 bacillomycin D(杆菌霉素D)。
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为进一步确定拮抗菌P-14产生的脂肽类拮抗物质,以菌株P-14的基因组DNA为模板扩增bacillomycin D合成的相关基因,其PCR产物的大小和理论值吻合,结果如图 4。将扩增得到的序列上传至NCBI进行同源性分析,比对结果表明菌株P-14中bamD基因片段与B. amyloliquefaciens FZB42 bacillomycin D基因同源性达99%。
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挥发性物质抑菌试验结果如图 5所示,接种3 d后,解淀粉芽孢杆菌处理中病原菌的菌落直径显著低于对照组,相对抑菌率为(26.19±3.82)%;此外,经过3次重复试验,在油茶健康叶片发病实验中,二分皿另一侧未接种P-14菌株时,油茶叶片发病严重,而接种P-14菌株的油茶叶片未发病(图 6),表明解淀粉芽孢杆菌的挥发性物质具有显著的抑菌活性。运用顶空固相微萃取法收集P-14菌株产生的挥发性气体进行GC-MS分析,可分离得到35种化合物,包含苯类物质(benzenes)10种、烷基类物质(alkyls)8种、醇类物质(alcohols)2种、酮类物质(ketones)11种、醛类物质(aldehydes)3种和1种酯类物质(esters),如表 1所示。
图 5 解淀粉芽孢杆菌的挥发性有机物质抑制病原菌生长(左边为处理,右边为对照)
Figure 5. B. amyloliquefaciens anti-fungal volatile activity on divided plates. Mycelial plug growth was inhibited in the presence of the bacteria streaked in the different compartments (right), compared to the control (left)
图 6 解淀粉芽孢杆菌的挥发性有机物质抑制叶片发病(左边为处理,右边为对照)
Figure 6. Volatile organic compounds of B. amyloliquefaciens reduce symptoms of Camellia oleifera leaf. Mycelial plug growth was inhibited in the presence of the bacteria streaked in the different compartments (right), compared to the control (left)
表 1 解淀粉芽孢杆菌P-14挥发性物质鉴定
Table 1. Volatile organic compounds produced by B. amyloliquefaciens P-14
苯类物质Benzenes 烷基类物质Alkyls 醇类物质Alcohols 酮类物质Ketones 醛类物质Aldehydes 酯类物质Esters 保留时间
Retention time化合物
Compounds保留时间
Retention time化合物
Compounds保留时间
Retention time化合物
Compounds保留时间
Retention time化合物
Compounds保留时间
Retention time化合物
Compounds保留时间
Retention time化合物
Compounds1.25 1, 4-Pentadiene 12.64 Cyclooctane, methyl- 10.48 Benzyl Alcohol 7.68 2-Heptanone 9.16 Benzaldehyde 14.51 n-pentadecyl ester 9.54 Phenol 13.05 Dodecane 10.35 1-Hexanol, 2-ethyl- 8.96 2-Heptanone, 6-methyl- 10.64 Benzaldehyde, 2-hydroxy- 10.09 Benzene, 1, 2-dichloro- 15.48 Cyclodecane 10.79 2-Nonanone 15.97 Dodecanal 11.21 Pyrazine, 2-ethyl-3, 5-dimethyl- 15.83 Tetradecane 11.04 Acetophenone 13.57 Benzothiazole 16.68 2-Methyl-1-dodecene 11.39 2-Nonanone 14.75 Pyrazine, 2, 5-dimethyl-3-(3-methylbutyl)- 16.79 Cyclododecane 12.40 2, 4, 6-Cycloheptatrien-1-one 15.24 Quinazoline, 4-methyl- 19.52 Eicosane 13.47 Benzofuran, 2-ethenyl- 16.25 Naphthalene, 2, 6-dimethyl- 20.06 Nonadecane 13.90 2-Undecanone 17.00 9-Octadecene, (E)- 15.3 4-Dodecanone 17.35 Butylated Hydroxytoluene 16.60 2-Tridecanone 17.84 2-Hexadecanone
油茶炭疽病拮抗细菌P-14的拮抗物质分析
Analysis of Metabolic Antagonists Against Colletotrichum camellia by Antagonistic Bacteria P-14
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摘要:
目的 探索解淀粉芽孢杆菌P-14对油茶炭疽病病原菌的拮抗物质,以期为油茶炭疽病的生物农药开发利用提供理论依据。 方法 利用酸沉淀法和高效液相色谱法分离解淀粉芽孢杆菌P-14发酵液中的拮抗物质,通过顶空固相微萃取法和气相色谱-质谱法对解淀粉芽孢杆菌P-14的挥发性物质进行了分离鉴定。 结果 分离后的组分a对油茶炭疽病的病原菌具有良好的抑制作用,其主要成分为C15 bacillomycin D(杆菌霉素D)。此外,共分离得到35种挥发性化合物,其相对抑菌率为(26.19±3.82)%,其中包含苯类物质(benzenes)10种、烷基类物质(alkyls)8种、醇类物质(alcohols)2种、酮类物质(ketones)11种、醛类物质(aldehydes)3种和1种酯类物质(esters)。 结论 解淀粉芽孢杆菌P-14能产生对油茶炭疽病的病原菌具有明显的拮抗作用的物质,并确定了其拮抗物质种类,为油茶炭疽病的生物农药开发提供了可靠的研究内容 Abstract:Objective The purpose of this study is to explore the metabolic antagonists against Colletotrichum camellia produced by antagonistic bacteria P-14, in order to provide a theoretical basis for the development and utilization of biopesticides of Camellia oleifera. Method The acid precipitation and high-performance liquid chromatography was used to identify the lipopeptide antibiotics produced by strain P-14, and the volatile compounds were analyzed by gas chromatography-mass spectrometry. Result The component a containing C15 bacillomycin D showed the strongest inhibitory effect against Colletotrichum camellia. In addition, a total of 35 volatile compounds were separated, the relative bacteriostatic rate was (26.19±3.82)%, including 10 benzenes, 8 alkyls, 2 alcohols, 11 ketones, 3 aldehydes, and 1esters. Conclusion Bacillus amyloliquefaciens P-14 could produce metabolic antagonists against Colletotrichum camellia, and the metabolic antagonists has been identified, which can provide with reliable research data for the development of biopesticides against C. oleifera. -
表 1 解淀粉芽孢杆菌P-14挥发性物质鉴定
Table 1. Volatile organic compounds produced by B. amyloliquefaciens P-14
苯类物质Benzenes 烷基类物质Alkyls 醇类物质Alcohols 酮类物质Ketones 醛类物质Aldehydes 酯类物质Esters 保留时间
Retention time化合物
Compounds保留时间
Retention time化合物
Compounds保留时间
Retention time化合物
Compounds保留时间
Retention time化合物
Compounds保留时间
Retention time化合物
Compounds保留时间
Retention time化合物
Compounds1.25 1, 4-Pentadiene 12.64 Cyclooctane, methyl- 10.48 Benzyl Alcohol 7.68 2-Heptanone 9.16 Benzaldehyde 14.51 n-pentadecyl ester 9.54 Phenol 13.05 Dodecane 10.35 1-Hexanol, 2-ethyl- 8.96 2-Heptanone, 6-methyl- 10.64 Benzaldehyde, 2-hydroxy- 10.09 Benzene, 1, 2-dichloro- 15.48 Cyclodecane 10.79 2-Nonanone 15.97 Dodecanal 11.21 Pyrazine, 2-ethyl-3, 5-dimethyl- 15.83 Tetradecane 11.04 Acetophenone 13.57 Benzothiazole 16.68 2-Methyl-1-dodecene 11.39 2-Nonanone 14.75 Pyrazine, 2, 5-dimethyl-3-(3-methylbutyl)- 16.79 Cyclododecane 12.40 2, 4, 6-Cycloheptatrien-1-one 15.24 Quinazoline, 4-methyl- 19.52 Eicosane 13.47 Benzofuran, 2-ethenyl- 16.25 Naphthalene, 2, 6-dimethyl- 20.06 Nonadecane 13.90 2-Undecanone 17.00 9-Octadecene, (E)- 15.3 4-Dodecanone 17.35 Butylated Hydroxytoluene 16.60 2-Tridecanone 17.84 2-Hexadecanone -
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