0 引言
【研究意义】烟草黑胫病（病原菌Phytophthora parasitica var. nicotianae）和赤星病（病原菌Alternaria alternata）是威胁世界烟草生产的两个重要病害。黑胫病常导致植株萎蔫坏死、根部变黑腐烂,最终整株死亡,每年对世界烟草生产造成百亿美元的损失[1]。目前,黑胫病的防治措施主要包括健康栽培、抗性品种利用、合理轮作及杀菌剂甲霜灵（metalaxyl）的施用。然而,长期大量施用甲霜灵会带来环境问题并产生Phytophthora抗甲霜灵菌株的风险[2]。赤星病是烟草成熟期叶部重要的病害之一,气候适宜往往暴发流行,造成严重经济损失。使用化学杀菌剂是赤星病防治的主要措施,但可能会导致烤后烟叶农残超标。因此,寻找能持续防控此病害和其他作物同类病害的新途径具有重要意义。【前人研究进展】利用有益微生物（生物农药）作为合理而安全的作物管理措施是较有前景的方法[3]。基于拮抗微生物和病原物互作的生物防治被认为是减少化学农药使用和改善植物健康的可替代或补充的方式[4,5]。研究表明,生物防治因子（biological control agents,BCAs）如芽孢杆菌（Bacillus）、假单胞菌（Pseudomonas）、放线菌（Streptomyces）、木霉菌（Trichodermas）和酵母菌（yeast）在抑制病原细菌和真菌方面均具有良好的表现[6,7,8,9,10]。生物防治因子的作用机制主要包括抗生作用,定殖位点、营养或矿物质的竞争,寄生和噬菌体[11];其中通过分泌抗真菌代谢物质（antifungal metabolites,AFMs）来发挥对病原真菌的抗生作用是最常见的策略,目前研究较多的抗真菌代谢物质主要有抗生素、酶和挥发物[12]。作为抗真菌代谢物质之一的混合着各种以碳为基础的气相混合物的挥发性有机物（volatile organic compounds,VOCs）,由于其小分子,体积小,易扩散到大气和土壤中,是理想的信息素,并且已被应用于昆虫、致病和污染有害菌的生物防治。作为生物防治因子的芽孢杆菌属细菌种类繁多,能够产生大量广谱且具有抗生活性的次生代谢产物,其中环状的脂肽类物质具有抗真菌和细菌等生物学功能[13],在这些抗生物质中,挥发性物质大都是由一组低分子量亲脂类物质组成的混合物,来源于很多微生物的不同生物合成途径,其中的一些挥发性代谢产物可采用气体处理的方式用于生物熏蒸[14]。围绕芽孢杆菌属细菌产生挥发物的抗生活性已展开了广泛的研究,20世纪90年代,FIDDAMAN等[15]报道了枯草芽孢杆菌（B. subtilis）分泌抗真菌挥发性物质过程中培养基质的重要性。近来又相继报道了解淀粉芽孢杆菌（B. amyloliquefaciens）在果实病害防治中的应用,解淀粉芽孢杆菌CPA-8产生的挥发物质抑制甜樱桃采后果实腐烂真菌核果褐腐菌（Monilinia laxa）、美澳型核果褐腐菌（M. fructicola）和灰葡萄孢（Botrytis cinera）菌丝的生长,能减轻樱桃采后腐烂程度。通过SPME-GC分析,CPA-8产生的主要挥发物为1,3-戊二烯、3-羟基丁酮和噻吩,各纯品的体外菌丝生长抑制试验和对3种腐烂病菌的EC50测定结果显示,噻吩是挥发物中最有效的成分[16]。在防治植物细菌病害方面,解淀粉芽孢杆菌T-5菌株产生的挥发物能够明显地抑制引起番茄青枯病的青枯雷尔氏菌（Ralstonia solanacearum）,且对该病菌的运动性、根部定殖、生物膜形成及其胞外多糖的产生具有抑制作用[17]。【本研究切入点】分离自贵州烟区黑胫病田块健康烟株根际土壤的短小芽孢杆菌（B. pumilus）AR03菌株,对烟草黑胫病菌、赤星病菌、青枯病菌、白粉病菌和炭疽病菌均有较好的拮抗活性,兼具促生作用[18,19,20,21]。进一步的研究表明,AR03发酵液（3×108 cfu/mL）对上述病原菌具有拮抗活性,但其除菌上清液却无活性。笔者对该拮抗菌株分泌的活性物质进行分析,发现该菌株能够产生挥发性物质且具有拮抗活性。【拟解决的关键问题】分析短小芽孢杆菌AR03菌株产生的挥发性物质对烟草黑胫病菌、赤星病菌的抑菌活性及其组成成分,为该菌株在病害生物防治上的应用提供理论依据。1 材料与方法
试验于2016年9月至2017年9月在中国农业科学院烟草研究所完成。1.1 供试菌株、培养基、培养条件与试剂
短小芽孢杆菌AR03（CGMCC No. 4117）分离和鉴定参见文献[18]。常规活化采用NA培养基（酵母提取物5.0 g,蛋白胨5.0 g,氯化钠1.5 g,琼脂13.0 g,蒸馏水1 L,pH 7.3,121℃灭菌20 min）,28℃恒温、黑暗条件培养36 h。将AR03接种于NB培养基（配方同NA,不加琼脂）中,120 r/min、28℃振荡培养48 h后获得AR03发酵液,经低温冷冻离心后的菌体经适量ddH2O梯度稀释后,采用分光光度计调整菌体悬浮液浓度为108 cfu/mL,备用。烟草黑胫病菌和烟草赤星病菌为笔者实验室在田间病株采集、分离、鉴定并保存。活化黑胫病菌和赤星病菌培养基分别为燕麦培养基（OA）和马铃薯葡萄糖培养基（PDA）,28℃培养5 d后备用。皮氏培养液：硝酸钙0.4 g,磷酸二氢钾0.15 g,硝酸镁0.15 g,氯化钙0.06 g,蒸馏水1 L,121℃ 15 min灭菌,备用。土壤浸出液：称取烟草品种红花大金元根际土壤25 g,溶于50 g蒸馏水中,搅拌均匀后室温静置30 min,经滤纸过滤后的溶液121℃ 15 min灭菌,备用。手动萃取手柄,50/30 μm DVB/CAR/PDMS萃取头,20 mL棕色顶空瓶,购自美国Supelco公司;1-十五烯、正构烷烃（C7-C30）购自北京百灵威公司;二氯甲烷、甲醇均为色谱纯;美国安捷伦7890B-5975C气相色谱质谱仪。
1.2 挥发性有机物对烟草黑胫病菌和赤星病菌体外抑制作用测定
1.2.1 对菌丝生长和形态的影响 接种环粘取活化的AR03菌落后均匀、密集地于NA平板上划线,使该细菌布满平板;另取烟草黑胫病菌和赤星病菌菌碟（Φ=5 mm）分别接种于OA和PDA平板的中央,参照王静等[21]方法在距菌碟1.5 cm处30°倾斜插入灭菌的盖玻片,然后与布满AR03菌体的NA平板对扣,周围用封口膜密封,28℃、黑暗条件下恒温培养,另以空白NA平板为对照,间隔2 d分别观察并测量处理和对照平板菌落直径,一直到对照平板菌丝长满,共调查4次,每处理3次重复。生长抑制率计算公式参照周翠等[22],比较挥发物的拮抗效果。试验重复2次。抑制率计算公式：抑制率（%）=（单独培养菌落直径-菌落趋向直径）/单独培养菌落直径×100
分别将培养6 d的烟草黑胫病菌和赤星病菌培养皿内附着菌丝的盖玻片置于倒置显微镜下（目镜10
倍）观察经挥发物处理后菌丝的形态变化。
1.2.2 对孢子萌发的影响 首先制备烟草黑胫病菌游动孢子悬浮液和赤星病菌分生孢子悬浮液。分别将预先活化的黑胫病菌和赤星病菌菌碟（Φ=5 mm）接种于OA和PDA平板中央28℃培养14 d后,诱导黑胫病菌产生孢子囊和游动孢子悬浮液制备方法如下：在盛有15 mL皮氏培养液无菌培养皿内接入8个黑胫病菌菌碟（Φ=6 mm）,然后加入100 μL土壤浸出液,置于28℃恒温箱中黑暗培养3 d后,将培养皿置于4℃搁置2 h,再转到28℃条件培养18 h,在无菌条件下,将培养皿内的菌饼移入盛有适量无菌水的离心管中,轻微振荡后经4层无菌纱布过滤收集游动孢子,载玻片法在光学显微镜下观察,随后通过梯度稀释和血小球计数板计数法将游动孢子悬浮液调整为2×104个/mL备用。赤星病菌孢子悬浮液制备方法如下：倒入适量的无菌水于长满菌丝的平板内,用涂布器涂布平板表面,使得菌丝和孢子悬浮于无菌水中,将此混合液经4层无菌纱布过滤后并收集,按照上述方法在倒置显微镜下观察并调整孢子悬浮液浓度为2×104个/mL,备用。
各取100 μL黑胫病菌游动孢子悬浮液和赤星病菌孢子悬浮液置于灭菌的凹玻片上,同时置于保湿的培养皿内,上面倒扣均匀划满AR03菌体的NA平板,对照为NA培养基,封口膜密封后置于28℃恒温分别黑暗培养6 h后,在倒置显微镜下观察形态变化。每个视野观察10个孢子,共观察5个视野,芽管的长度是孢子的两倍视为孢子萌发。计算各处理抑制游动孢子和分生孢子萌发和生长的抑制率,每处理3次重复。试验重复2次。孢子萌发抑制率计算公式：
孢子萌发抑制率（%）=（对照组孢子萌发数-处理组孢子萌发数）/对照组孢子萌发数×100
1.2.3 挥发性有机物离体叶片的抑菌测定 挥发物抑制烟草黑胫病和赤星病的离体叶片生测接种采用菌饼接种和孢子悬浮液悬滴接种,具体如下：于NA平板（直径15 cm）密集划线接种AR03菌株,对照为空白的NA平板;另外培养皿底部分别放置2片大小适宜的烟草叶片红花大金元（用于黑胫病生测试验）和品种NC89（成熟期,用于赤星病抗病试验）,浸湿的棉球包围叶柄保湿,叶片表面无菌水喷雾后,在主脉两侧各选取适宜数量的接种点,针刺制造伤口后,分别接种黑胫病菌碟（Φ=6 mm）和悬滴100 μL赤星病菌孢子悬浮液（浓度为1×107个孢子/mL）,每处理10个皿,3次重复。将接种细菌的培养皿与接种叶片的培养皿对扣后Parafilm封口膜密封,30℃黑暗条件下对峙培养40 h和80 h后调查病斑在叶片上扩展情况并计算抑制率。抑制率计算公式：
病斑扩展抑制率（%）=（对照组病斑直径-处理组病斑直径）/对照组病斑直径×100
1.3 挥发性有机物的收集及其组分分析
1.3.1 1-十五烯内标溶液配制 称取20 mg 1-十五烯对照品,纯度不低于98.5%,用甲醇定容于10 mL容量瓶中,得到浓度为2 mg·mL-1 1-十五烯内标母液,然后取适量1-十五烯内标母液用甲醇稀释400倍,得到浓度为5 μg·mL-1 1-十五烯内标溶液,备用。1.3.2 AR03细菌挥发物收集-顶空-固相微萃取法 移液器吸取3 mL尚未冷却的NA培养基（约55℃）,倾倒于20 mL灭菌的棕色顶空瓶中,并将瓶体倾斜15°角,待培养基冷却凝固后,取100 μL预先制备好的AR03菌悬液均匀涂布于培养基斜面上,迅速用含有聚四氟内涂层的瓶盖封口,将顶空瓶置于28℃恒温箱中静置、黑暗培养,为了排除培养基释放的挥发物和萃取头涂层流失对细菌挥发物鉴定结果的干扰,设置不涂布AR03菌悬液处理为空白对照,每个样品3个重复。
将培养2 d的顶空瓶置于室温中平衡20 min后,采用50/30 μm DVB/CAR/PDMS萃取头（购于Supelco公司）进行采样,萃取头使用前,需先按照厂家提供的说明书进行老化。采样前首先用5 μL尖头微量注射器向顶空瓶侧壁注入2 μL浓度为5 μg·mL-1 1-十五烯内标溶液,注意尽量避免接触培养基基质,平衡30 min后,将SPME针管刺入顶空瓶,同时将萃取头推出,置于顶空瓶上部1/3处,采集细菌分泌的挥发性有机物,采样时间为30 min。采集完毕后,将萃取头收起,并将针管拔出样品瓶,样品收集结束。
1.3.3 利用气象色谱-质谱联用（GC-MS）技术分析挥发性物质组分 GC-MS分析条件：色谱柱DB-5MS弹性毛细管色谱柱,规格为30 m×0.25 mm×0.25 μm;进样口250℃：不分流模式;初始温度40℃,以100℃·min-1速率升至250℃,解吸时间5 min;载气为99.999%氦气,流速为1.0 mL·min-1;柱温箱：初始温度40℃,保持2 min,以5℃·min-1速率升至180℃,保持10 min,以10℃·min-1速率升至280℃,保持15 min,总运行时间为55 min;EI离子源,电子能量70 ev;离子源温度230℃,四级杆温度150℃,传输线温度280℃;全扫描模式,扫描范围30—400 m/z。数据采集处理系统,安捷伦MassHunter（B.05.02）软件。
1.3.4 抑菌挥发性有机物成分鉴定 挥发物MS结果经NIST和WILIY谱库自动检索,要求匹配率>80,初步进行识别。对于识别的每种代谢物,根据其相邻正构烷烃的保留时间和碳原子数,计算其保留指数,然后查阅文献报道的该物质在相同色谱柱中的保留指数（retention index,RI）,进一步进行确证。保留指数计算公式：
RI=100z+100[TR(x)-TR(z)]/[TR(z+1)-TR(z)]（线性程序升温）
式中,TR(x)、TR(z)、TR(z+1)分别代表组分x及碳数为z、z+1正构烷的保留温度。且TR(z)<TR(x)<TR(z+1),由于保留温度和保留时间通常具有高度的相关性,所以用保留时间代替上式中的保留温度来进行计算保留指数。
1.3.5 不同生长期抑菌挥发性有机物组分定量分析 按照1.3.2方法分别将培养1、3、6 d的AR03,按照1.3.2、1.3.3和1.3.4方法收集细菌挥发物进行GC-MS检测,并进行定量分析。抑菌挥发性有机物半定量分析以添加到顶空瓶中的1-十五烯作为内标（internal standard,IS）,假设内标与生防菌株AR03挥发物的相对校正因子为1,按下述公式计算目标代谢物的相对含量。
mi= m15×（Ai/A15）
式中,mi为目标代谢物含量（μg）;m15为加入1-十五烯的量（μg）;Ai为目标代谢物总离子流图峰面积;A15为1-十五烯总离子流图峰面积。
2 结果
2.1 挥发性有机物对供试病原菌菌丝生长的影响
平板对扣法测定结果显示,挥发性有机物对烟草黑胫病菌和赤星病菌菌丝生长的抑制作用较强,对赤星病菌的抑制表现为菌落生长缓慢且培养性状异常;对黑胫病菌的抑制表现为菌丝生长稀疏且菌落生长迟缓,而对照菌落生长正常（图1-A、1-B）。培养6 d的黑胫病菌和赤星病菌菌丝生长速率的测定结果如图2。从图中可以看出,该抑菌挥发物对两种供试病原菌菌丝生长均有不同程度的抑制作用,抑制率随时间的延长而增长,对峙培养8 d时,对黑胫病菌和赤星病菌的菌丝生长抑制率分别为67.04%和70.00%。镜检观察显示,挥发物处理后的黑胫病菌生长缓慢、稀疏,菌丝扭曲、分枝增多且断裂;经挥发物处理的赤星病菌菌丝畸形,其上不产生分生孢子梗,内含物聚集成团导致菌丝粗细不均、缢缩。
显示原图|下载原图ZIP|生成PPT图1挥发性有机物对烟草赤星病菌（A）和黑胫病菌（B）的抑菌活性
-->Fig. 1Inhibitory activity of VOCs against A. alternata (A) and P. parasitica (B)
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显示原图|下载原图ZIP|生成PPT图2挥发性有机物对烟草赤星病菌和黑胫病菌菌丝生长的影响
-->Fig. 2Effect of VOCs on mycelial growth of A. alternata and P. parasitica
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2.2 挥发性有机物对供试病原菌孢子萌发的影响
赤星病菌菌分生孢子经抑菌挥发性有机物处理后萌发受到抑制,且延迟萌发后的芽管生长缓慢、短小畸形,大部分分生孢子畸形,表现为各分隔处的细胞肿胀成圆球状泡或其他不规则形,而对照孢子均正常萌发且芽管生长正常。经挥发性有机物处理后的黑胫病菌产生的孢子囊数量明显减少,游动孢子萌发延迟,且萌发后芽管比空白对照生长缓慢。2.3 挥发性有机物抑菌活性的离体叶片测定
挥发性有机物抑菌试验结果如表1和图3,培养至40 h和80 h,对照叶片的病斑扩散迅速,黑胫病发病率为92.50%,病斑表现为水浸状斑块沿接种点向周围扩散为近圆形,病斑直径平均为25.10 mm;经挥发物处理后叶片发病率为70.83%,病斑直径的平均值为9.45 mm,挥发物对黑胫病斑扩散的抑制率为62.35%。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%。
显示原图|下载原图ZIP|生成PPT图3AR03菌株挥发性有机物抑菌活性的离体叶片测定
-->Fig. 3Inhibitory activity of VOCs produced by strain AR03 in vitro leaves
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Table 1
表1
表1AR03菌株分泌的挥发性有机物对烟草黑胫病和赤星病的防治效果
Table 1Control effect against tobacco black shank and brown spot of VOCs produced by strain AR03
| 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 | — |
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2.4 基于SPME GC-MS技术获得AR03抑菌挥发性有机物总离子流图
将上述获得的吸附挥发物的萃取头,在全扫描模式下进行分析,得到正构烷烃（C10-C20）、空白样品挥发物和细菌挥发性有机物的总离子流图（图4-A、4-B、4-C）。根据前期试验结果,初步判定AR03产生的抑菌代谢混合物为倍半萜烯类物质（C15H24）,本方法选用结构式相近的1-十五烯为内标物（C15H30）,因其属性与目标代谢物相似,在色谱图中出峰时间比较接近,且经检验AR03抑菌挥发代谢物在十五烯的保留时间内,无干扰峰出现（图4-A）。将GC-MS采集的AR03挥发物总离子流图,扣除空白样品挥发物总离子流图中共有色谱峰（图4-B）,即为挥发性有机物特征指纹峰（图4-C）,该菌体分泌的抑菌挥发性有机物共为7种,色谱峰出现的时间相对集中于22—25 min,主要成分为保留时间24.72 min的一种化合物,相对含量很高,而其他各物质含量相对较低,且各挥发物间不存在重叠性。
显示原图|下载原图ZIP|生成PPT图4AR03菌株分泌抑菌挥发性有机物的SPME GC-MS分析
-->Fig. 4SPME GC-MS analysis of VOCs produced by strain AR03
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2.5 挥发性有机物的定性与定量分析
将2.4中获得细菌挥发物总离子流图,扣除空白样品挥发物总离子流图中共有色谱峰,即为AR03菌株释放的挥发性特征指纹峰。计算每个色谱峰的保留指数,结合使用NIST和WILIY谱库,对总离子图中色谱峰进行定性识别,设置匹配度阈值为80,AR03抑菌挥发代谢物定性匹配结果见表2,该菌株分泌的抑菌挥发物共7种且均为C15H24结构的倍半萜烯类化合物,根据色谱峰出现的先后顺序分别为二氢姜黄烯、(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;内标1-十五烯在色谱图中出峰时间比较接近,且经检验AR03抑菌挥发性有机物在十五烯的保留时间内。Table 2
表2
表2AR03菌株抑菌挥发性有机物的鉴定
Table 2Identification of VOCs produced by strain AR03
| 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 | — | — |
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2.6 不同生长期AR03挥发性有机物定量分析
AR03菌株分别培养1、3和6 d后,其分泌的抑菌挥发性有机物相对含量有一定差异（表3）,且随着时间的延长呈递减趋势,当细菌培养1 d时,挥发物各组分相对含量最高,尤其是β-倍半萜水芹烯的分泌量高达112.01 ng,占总含量的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%;培养至3 d时,除了二氢姜黄烯含量小幅度升高外,挥发物其他各组分相对含量均逐渐降低,培养至6 d时,相对含量减少50%左右,各组分含量下降较明显。Table 3
表3
表3AR03菌株挥发性有机物组分定量分析
Table 3Quantitative analysis of VOCs produced by strain AR03
| 名称 Name | 倍半萜烯代谢物含量 Relative contents of sesquiterpenes (ng) | ||
|---|---|---|---|
| 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 |
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3 讨论
能分泌抑菌挥发性有机物的细菌在微生物界普遍存在,ZOU等[23]随机选择1 018种细菌中,有328株细菌可以产生抗真菌的挥发性物质。与非挥发性抗菌物质相比,挥发性抑菌物质分子小,更易于在土壤和环境中渗透和扩散,能更全方位地杀灭环境中的病原菌,并能通过调节激素的代谢促进植物生长[24]。在植物病害生物防治中,细菌分泌抗真菌性挥发物质的鉴定及其应用是由FERNANDO等[25]首次报道,分离自大豆根部的绿针假单胞菌（Pseudomonas chlororaphis）PA-23挥发物在体外和土壤中能抑制核盘菌（Sclerotinia sclerotiorum）菌丝生长及子囊孢子和菌核的萌发,凹玻片萌发法测定结果显示挥发物对子囊孢子萌发的抑制率为54%—90%;本研究中的短小芽孢杆菌AR03菌株产生的挥发物对烟草黑胫病菌和赤星病菌菌丝、菌落生长和孢子萌发具有不同程度的抑菌和致畸作用,结合AR03菌株先前的研究结果分析其活性物质,该菌株产生胞外抑菌挥发物与其拮抗活性紧密相关,且是该菌株对黑胫病菌和赤星病菌拮抗机制的关键因子。挥发性有机物是很理想的信息化学物质,由于它们的易扩散性,挥发物的活动范围可以从近处的互作扩展到更远的距离[26]。本试验中,AR03菌株分泌的抑菌挥发物利用其扩散的特性,抑制了离体叶片上病斑的扩展,从而减轻病害发生。在烟草生产上,可以将菌株施入土壤或喷施在叶片上,产生的抑菌挥发物在土壤或烟株周围的环境中扩散进而充分保护烟株根部或叶片免受病原菌的侵染。细菌在代谢过程中产生的挥发性物质种类较多,KAI等[11]通过顶空收集法和GC-MS技术分析了不同细菌产生的挥发性物质,发现不同的细菌能分别产生1—30种不同组分的挥发性化合物。已有很多研究报道了关于挥发性有机物具有抗植物病原真菌的活性,例如三甲胺（trimethylamine）抑制白地霉（Geotrichum candidum）菌丝生长和孢子形成[27],ALMENAR等[28]鉴定了Bacillus spp.产生的14种不同的挥发物,包括二乙基己醇（2-ethyl-hexanol）、对羟基苯甲醛（4-hydroxybenzaldehyde）和壬酮（2-nonanone）等抗真菌挥发物;ARREBOLA等[29]采用GC-MS技术检测分析拮抗果实采后病菌青霉（Penicillium crustosum）的枯草芽孢杆菌PPCB01和解淀粉芽孢杆菌PPCB04产生挥发性抑菌物质,PPCB01菌株产生21种不同类型的挥发物,而PPCB04菌株产生8种不同的挥发物,其中3-羟基-2-丁酮是主要抑菌成分,含量分别为45.98%和97.52%。在本研究中,采用将AR03菌株产生的挥发性有机物经顶空收集采样并进行GC-MS定性和定量分析,省去了普遍采用有机溶剂萃取的步骤,避免了有机溶剂不能完全萃取到所有挥发物质的缺点。GC-MS分析表明,AR03菌株产生7种C15H24结构的倍半萜烯类物质。相关研究表明,大多数倍半萜烯化合物具有芳香和重要的生物活性,是研究天然产物和开发新药物的重要来源,广泛存在于生姜（Zingiber officinale）[30]、人参（Panax ginseng）[31]、刺芹属植物Eryngium caucasicum [32]及中药假鹰爪（Desmos chinensis）果实[33]等植物体中,本研究报道了来源于微生物次级代谢产物的倍半萜烯类化合物,为挖掘和开发具有药用前景的倍半萜烯提供了新的微生物资源和途径。
天然产物在开发抗癌药物中发挥着重要的作用,约80%的药物来源于天然产物[34]。已有相关文献报道本试验中抑菌挥发物质的一些组分具有重要的生物活性,相对含量为80.64%的β-倍半萜水芹烯是一种重要的香料和药理活性物质,1984年首次发现于生长于冰岛的早花百里香的挥发油中[35];还存在于姜、艾草（Artemisia annua）[36]及美国冷杉（Amabilis fir）叶片和嫩枝的挥发油中[37]。近期,TYAGI等[38]从姜黄粉的酒精浸提物中分离获得的β-倍半萜水芹烯具有抗恶性细胞扩散的活性,能抑制人类白血病细胞、骨髓瘤细胞和结直肠癌细胞的增殖;通过细胞内酯酶活性、质膜完整性和细胞相试验发现,该物质还可以高效地抑制癌细胞克隆形成和诱导细胞凋亡。另外一个相对含量较高的组分(E)-β-金合欢烯是一种重要的警报信息素,大多数蚜虫都能产生和利用该种激素,它的挥发释放参与蚜虫间的化学聚集,特别是能干扰蚜虫取食,可用于蚜虫的生物防治[39,40]。AR03菌株挥发物中的α-姜烯也具有重要的药理活性,据BOU等[41]研究发现,从林生脚骨脆叶（Casearia sylvestris）粗提精油中分离的α-姜烯纯品对人宫颈癌HeLa、U-87、Siha和HL60细胞系具有细胞毒素活性,但其抑制中浓度（IC50）比粗提精油高。本研究鉴定的倍半萜烯各组分的药理活性尚需进一步验证。
生物挥发性物质的释放是动态的过程,在细菌生长的过程中,随着时间的变化和培养基的消耗,挥发物质的种类和数量会发生变化[42,43]。本研究通过保留指数和以1-十五烯为内参测定AR03菌株不同培养时间产生挥发物的相对含量,发现随着时间的延长,各组分种类不变,但相对含量呈降低趋势,原因主要包括以下两个方面：其一可能是细菌生长经过对数期、稳定期进入衰亡期,繁殖越来越慢,代谢活动减慢,代谢产物亦随之减少;另一个可能原因是GC-MS分析挥发物除了倍半萜物质外,还含有戊二烯（C5H8）,由于戊二烯含有含多种同分异构体（1,3-戊二烯、反式-1,3-戊二烯、1,4-戊二烯、反式-1,4-戊二烯等）,这些异构体的质谱图相似度极高,且在DB-5MS色谱柱中保留行为较差,出峰时间较早（在本文报道色谱条件下1.6 min出峰）,无法准确确证。因此笔者推测挥发物中的倍半萜烯类物质具有化学不稳定性,随着培养时间的延长,可能降解为单体戊二烯,加上该物质无生物活性,本试验未对其进行分析。另外值得注意的是,先期的研究结果显示,AR03发酵液对烟草赤星病菌分生孢子萌发有很强的抑制效果,经一定浓度含菌发酵液处理的分生孢子几乎不萌发,而本试验结果显示,AR03产生挥发物对供试病原菌分生孢子萌发的抑制和致畸作用比发酵液的抑菌作用弱,因此,挥发物质的抑菌活性可能只是AR03菌株抑菌作用的一部分;另外,挥发物对分生孢子萌发的影响采用的凹玻片萌发法,因为分生孢子分散于无菌水中,挥发物无法直接作用于分生孢子从而不能充分发挥其抑菌活性;AR03菌株分泌的挥发物在密封的培养皿空间内分散后浓度低可能也是影响其抑菌活性的重要因素。
4 结论
经室内培养皿对扣熏蒸法、凹玻片萌发法和离体叶片抗病测定,基本确定短小芽孢杆菌AR03菌株分泌的挥发性有机物对烟草黑胫病菌和赤星病菌菌丝生长具有抑制和致畸作用,抑制黑胫病菌产生孢子囊并延迟游动孢子的萌发;抑制赤星病菌分生孢子的萌发并引致孢子畸形;同时抑制黑胫病菌和赤星病菌病斑在叶片上的扩展。SPME GC-MS分析表明,AR03能分泌二氢姜黄烯、(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7种C15H24结构的倍半萜烯类物质,其中β-倍半萜水芹烯相对含量最高,其次为(E)-β-金合欢烯和α-姜烯。短小芽孢杆菌AR03菌株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。
The authors have declared that no competing interests exist.
参考文献 原文顺序
文献年度倒序
文中引用次数倒序
被引期刊影响因子
| [1] | . In order to identify tobacco ( Nicotiana megalosiphon) genes involved in broad-spectrum resistance to tobacco black shank ( Phytophthora parasitica var. nicotianae), suppression subtractive hybridization (SSH) was used to generate a cDNA from transcripts that are differentially expressed during an incompatible interaction. Forty-eight differentially expressed genes were selected, sequenced and analyzed. The cDNA collection comprised a repertoire of genes associated with various processes. Real-time PCR analysis of a subset of these genes confirmed the differential expression patterns between the compatible and incompatible interaction. The experiments demonstrated for the first time that hrs203J gene and RING finger protein gene exhibited strong induction during several incompatible interactions. Also, these genes were found not to be induced in compatible interactions. The set of differentially expressed tobacco genes associated to resistance could be exploited in strategies to develop durable resistance in cultivated tobacco plants. |
| [2] | |
| [3] | . Abstract Although the number of biocontrol products is increasing, these products still represent only about 1% of agricultural chemical sales. Yet these are important contributions because biocontrol agents offer disease management alternatives with different mechanisms of action than chemical pesticides. Trends in research include the increased use of biorational screening processes to identify microorganisms with potential for biocontrol, increased testing under semicommercial and commercial production conditions, increased emphasis on combining biocontrol strains with each other and with other control methods, integrating biocontrol into an overall system. |
| [4] | . |
| [5] | . The advances of plant and plant growth promoting bacteria (PGPB) interaction research focusing on the principles and mechanisms of action of PGPB, both free living and endophytic bacteria, and their use or potential use for the biological control of plant diseases are reviewed.. |
| [6] | |
| [7] | . The effectiveness of two Streptomyces spp. strains to controlpathogenic fungi was studied in stored maizegrain. The treatments included seeddisinfection and inoculation with Streptomyces spp. strains previously isolatedfrom maize rhizosphere. Actinomycete inoculumconsisted of filtered suspension and totalsuspension of fermentor-produced Streptomyces spp. strains biomass. Treatmentswith Streptomyces spp. strains aloneeffectively suppressed the development of Aspergillus spp., Curvularia lunata , and Drechslera maydis and significantly(p < 0,05) reduced the incidence of Fusarium subglutinans and Cephalosporiumacremonium . Among the inoculation treatments,nondisinfested seed inoculated with filteredsuspension was the only treatment that did notsuppress the development of Penicilliumspp. Maize seed inoculation with totalsuspension of strains was the most effectivetreatment to control the incidence of seedpathogenic fungi. The development of the Diplodia maydis was only suppressed by thecombination of seed disinfection andinoculation with total suspension of strains.Although, the strain DAUFPE 11470 showed thegreatest effectiveness for controlling thefungi pathogenic to seed, root and shootdevelopment was reduced by treatment with thisstrain.The results indicate that Streptomyces spp. strains reduce the incidenceof seed pathogenic fungi and have potential asa biological control agent. However, an efficient methodof seed treatment with the biological controlagent must be developed before it can become anagricultural practice. |
| [8] | Biocontrol agents generally do not perform well enough under field conditions to compete with chemical fungicides. We determined whether transgenic strain SJ3-4 of Trichoderma atroviride, which expresses the Aspergillus niger glucose oxidase-encoding gene, goxA, under a homologous chitinase (nag1) promoter had increased capabilities as a fungal biocontrol agent. The transgenic strain differed only slightly from the wild-type in sporulation or the growth rate. goxA expression occurred immediately after contact with the plant pathogen, and the glucose oxidase formed was secreted. SJ3-4 had significantly less N-acetylglucosaminidase and endochitinase activities than its nontransformed parent. Glucose oxidase-containing culture filtrates exhibited threefold-greater inhibition of germination of spores of Botrytis cinerea. The transgenic strain also more quickly overgrew and lysed the plant pathogens Rhizoctonia solani and Pythium ultimum. In planta, SJ3-4 had no detectable improved effect against low inoculum levels of these pathogens. Beans planted in heavily infested soil and treated with conidia of the transgenic Trichoderma strain germinated, but beans treated with wild-type spores did not germinate. SJ3-4 also was more effective in inducing systemic resistance in plants. Beans with SJ3-4 root protection were highly resistant to leaf lesions caused by the foliar pathogen B. cinerea. This work demonstrates that heterologous genes driven by pathogen-inducible promoters can increase the biocontrol and systemic resistance-inducing properties of fungal biocontrol agents, such as Trichoderma spp., and that these microbes can be used as vectors to provide plants with useful molecules (e.g., glucose oxidase) that can increase their resistance to pathogens. |
| [9] | |
| [10] | Aims: To develop a pilot-plant fermentation process for the production of the yeast Rhodotorula minuta , to be used as a biocontrol agent of mango anthracnose, using a low-cost culture medium. To develop a stable liquid formulation that preserve high viability of the yeast stored at 4°C. |
| [11] | |
| [12] | . |
| [13] | Contamination of plants and seeds with microorganisms is one of the main problems in the production and distribution of various agricultural products, as well as raw herbal material for the preparation of herbal remedies. In targeting microbial contamination, among other bacteria, Bacillus species showed a significant capacity for biocontrol. The antifungal activity of 14 isolates of Bacillus spp. against 15 fungal isolates from medicinal plants was examined utilizing a dual plate assay. The strongest and broadest antagonistic activity against all fungi tested was exhibited by isolates SS-12.6 and SS-13.1 (from a 43% to 74% reduction in fungal growth), while isolates SS-39.1 and SS-39.3 were effective against the fewest fungus species and also had the weakest antifungal activity. The effect of a crude lipopeptide extract (CLE) of Bacillus sp. SS-12.6 was similar to that achieved by a dual culture with isolate SS-12.6, confirming that the antagonism was the result of the antifungal activities of lipopeptides. In addition, essential oils of thyme (0.55mg/mL) and savory (0.32mg/mL) in various combinations with the CLE of SS-12.6 were tested for antifungal activity, and additive and synergistic effects for some of the fungi were obtained. When testing the effect of CLE, oils (0.40mg/mL for thyme oil and 0.21mg/mL for savory oil) and combinations in situ on marigold seeds, a reduction of total fungal infection without an adverse effect on germination was accomplished by 6-h treatments with CLE of SS-12.6 (85% reduction of fungal infection and 63% germination), supernatant from liquid culture of SS-12.6 (more than 90% reduction of fungal infection with 69% seed germination) and combinations of CLE and savory oil (77% reduction of fungal infection and 62% seed germination) and CLE with thyme and savory oils (about 75% reduction of fungal infection with 69% seed germination). |
| [14] | . Significant postharvest losses occur during the supply chain of fresh produce. Postharvest decay is one of the main factors that determines losses and compromises the quality of fruit and vegetables. Traditionally postharvest decay control is achieved using chemical fungicides; however, the important concerns relating to environmental and human health require the development of novel methods for the control of postharvest decay. Furthermore, the consumer demand and the purchasing power are higher for fresh produce that are free from pesticide application. This review highlights the research literature based information on the application of microbial and plant volatile organic compounds (VOCs) to control postharvest decay, their practical applications in postharvest systems and the future perspectives. The volatile nature of VOCs could be potentially employed with success as gaseous treatments in a process defined 鈥榖iofumigation鈥. Plant-produced volatiles including among others, aldehydes such as acetaldehyde, 2-E-hexenal and benzaldehyde, alcohols such as ethanol and acetic acid, essential oils (EOs) and isothiocyanates (ITCs) and microbial volatile organic compounds have been recognised as potential substances in preventing pathogenic infections in many horticultural commodities. However, some issues have to be well elucidated in order to admit these substances in a large-scale application to improve the competitiveness of the fresh produce at the quality stringent EU, USA, and the Japanese markets. The main concern are related to the registration process, but also VOCs degradation and residues in fruit, formulation and organoleptic impact, are aspects that have to be thoroughly considered prior to commercialisation of the selected VOC. Furthermore, VOCs could have an effective role for an eco-chemical approach in postharvest disease control since these biobased products, if compared to conventional ones, can offer clear environmental benefits due to their renewability, biodegradability and hypotoxicity. |
| [15] | An antibiotic-producing strain of Bacillus subtilis has been shown to produce potent antifungal volatiles (AFV). These volatiles are active against a range of fungal species and are produced on a range of growth media and in loam-based compost. In vitro antifungal volatile activity on nutrient agar is enhanced with the addition of D-glucose, complex carbohydrates and peptones. The addition of L-glucose led to significantly less AFV activity than comparable levels of D-glucose. Growth studies in liquid culture revealed that B. subtilis failed to grow in response to L-glucose. Further growth studies on solid media showed no clear correlation between enhanced bacterial growth and increases in in vitro AFV activity in response to supply of substrates. Low level AFV activity was also detected from oilseed rape roots inoculated with B. subtilis . Gas chromatography mass spectrometry headspace analysis of B. subtilis cultures grown on various substrates revealed common similarities between substrates promoting AFV activity, although it was not possible to isolate individual antifungal compounds. |
| [16] | 61VOCs from CPA-8 were tested against mycelial and colony growth ofM.02laxa,M.02fructicolaandB.02cinerea.61Three different growth media were used for CPA-8 VOCs production.61CPA-8 grown on TSA exhibited the highest percentage of mycelial growth inhibition.61VOCs emitted by CPA-8 were identified by SPME-GC and selected for EC50estimation.61CPA-8 VOCs and pure thiophene were evaluated on artificially inoculated cherry fruit. |
| [17] | |
| [18] | . . |
| [19] | . 为测定短小芽孢杆菌AR03对烟草黑胫病菌的拮抗作用及其发酵菌剂的田间防效，通过对峙培养法、固态发酵及田间小区试验的方法发现，AR03菌体和发酵液对烟草黑胫病菌具有很强的拮抗活性，而其除菌上清无拮抗活性，初步说明AR03分泌的抗菌物为胞内物质。AR03经固态发酵后的活菌数为3．82×10^11cfu／g。该菌剂的田间小区试验结果显示，对混合有青枯病发生的烟草黑胫病的防治效果明显好于对照药剂，且与72％甲霜灵·锰锌可湿性粉剂协同防治效果最好。因此，AR03对烟草黑胫病和青枯病具有可持续控病作用，是1株极具开发潜力的生防菌株。 为测定短小芽孢杆菌AR03对烟草黑胫病菌的拮抗作用及其发酵菌剂的田间防效，通过对峙培养法、固态发酵及田间小区试验的方法发现，AR03菌体和发酵液对烟草黑胫病菌具有很强的拮抗活性，而其除菌上清无拮抗活性，初步说明AR03分泌的抗菌物为胞内物质。AR03经固态发酵后的活菌数为3．82×10^11cfu／g。该菌剂的田间小区试验结果显示，对混合有青枯病发生的烟草黑胫病的防治效果明显好于对照药剂，且与72％甲霜灵·锰锌可湿性粉剂协同防治效果最好。因此，AR03对烟草黑胫病和青枯病具有可持续控病作用，是1株极具开发潜力的生防菌株。 |
| [20] | . The inhibitory effects of Bacillus pumilus AR03 strain on tobacco anthracnose (Colletotrichum destructivum) were evaluated by culture assay and pot experiments in this study. The results showed that the AR03 strain had a good inhibitory effect on C.destructivum in both culture assay and pot experiments. The fermentation broth of AR03 (1脳107 cfu/mL) could significantly inhibit the mycelium growth and spore germination. Pot test effect of AR03 bacterial suspension (1脳108 cfu/mL) was 83.03%, well matched with the protective effect of 25% triadimefon WP. The strain AR03 is a broad-spectrum, and efficient biocontrol strain. The inhibitory effects of Bacillus pumilus AR03 strain on tobacco anthracnose (Colletotrichum destructivum) were evaluated by culture assay and pot experiments in this study. The results showed that the AR03 strain had a good inhibitory effect on C.destructivum in both culture assay and pot experiments. The fermentation broth of AR03 (1脳107 cfu/mL) could significantly inhibit the mycelium growth and spore germination. Pot test effect of AR03 bacterial suspension (1脳108 cfu/mL) was 83.03%, well matched with the protective effect of 25% triadimefon WP. The strain AR03 is a broad-spectrum, and efficient biocontrol strain. |
| [21] | . <p>采用对峙培养法、凹玻片法、LB琼脂培养基萌发法测定短小芽孢杆菌AR03对烟草赤星病菌和白粉病菌的抑制作用.结果表明： AR03菌液对2种病菌的菌丝生长和分生孢子萌发均有明显的拮抗作用；对赤星病菌的抑制作用表现为:经AR03菌液原液(3×10<sup>8</sup>cfu·mL<sup>-1</sup>)处理的菌丝隔间变短、肿胀且集结成团，内含物聚集，菌丝顶端生长膨大畸形；经该菌液处理的赤星病菌分生孢子不萌发或萌发产生畸形芽管，分生孢子变形、肿大，纵横分隔部分的组织膨胀呈泡状.AR03菌液原液、30倍液和200倍液对白粉病菌分生孢子萌发的平板抑制率分别为100%、91.4%和 69.3%，菌液对分生孢子萌发的破坏作用表现为分生孢子不萌发，细胞肿胀变形、细胞原生质解体或收缩，孢子内、外壁分离，由于原生质外泄，一些分生孢子内部呈中空状.温室防治试验结果表明： 不同浓度AR03菌悬液处理对烟草白粉病的防治效果存在显著差异，第二次药后7和15 d，AR03菌液原液的防治效果分别达到83.8%和90.3%，与对照药剂差异不显著；而100倍稀释液的防效分别为70.0%和73.3%，与对照药剂差异显著.AR03菌株防治白粉病的持效期为30 d以上.</p> <p>采用对峙培养法、凹玻片法、LB琼脂培养基萌发法测定短小芽孢杆菌AR03对烟草赤星病菌和白粉病菌的抑制作用.结果表明： AR03菌液对2种病菌的菌丝生长和分生孢子萌发均有明显的拮抗作用；对赤星病菌的抑制作用表现为:经AR03菌液原液(3×10<sup>8</sup>cfu·mL<sup>-1</sup>)处理的菌丝隔间变短、肿胀且集结成团，内含物聚集，菌丝顶端生长膨大畸形；经该菌液处理的赤星病菌分生孢子不萌发或萌发产生畸形芽管，分生孢子变形、肿大，纵横分隔部分的组织膨胀呈泡状.AR03菌液原液、30倍液和200倍液对白粉病菌分生孢子萌发的平板抑制率分别为100%、91.4%和 69.3%，菌液对分生孢子萌发的破坏作用表现为分生孢子不萌发，细胞肿胀变形、细胞原生质解体或收缩，孢子内、外壁分离，由于原生质外泄，一些分生孢子内部呈中空状.温室防治试验结果表明： 不同浓度AR03菌悬液处理对烟草白粉病的防治效果存在显著差异，第二次药后7和15 d，AR03菌液原液的防治效果分别达到83.8%和90.3%，与对照药剂差异不显著；而100倍稀释液的防效分别为70.0%和73.3%，与对照药剂差异显著.AR03菌株防治白粉病的持效期为30 d以上.</p> |
| [22] | . 初步研究了分离自石榴果实中的一株枯草芽孢杆菌Bacillus subtilis所产生的挥发性物质对几种常见林果病原菌的抑菌作用。发现该挥发性物质对不同病原菌菌丝生长具有一定的抑制作用,其中对杨树溃疡病菌 Botryosphaeria dothidea的抑制率达60%。将不同病原菌株与该挥发性物质互作后的菌丝形态与正常菌丝进行比较,结果表明：处理后的杨树溃疡病菌、石榴疮痂病菌 B.dothidea、苹果轮纹病菌B.dothidea及葡萄白腐病菌Coniothyrium diplodiella的菌丝分支增多,扭曲,菌丝顶端和中部细胞膨大;杨树腐烂病菌Valsa sordida及桑紫纹羽病菌Helicobasidium purpureum的菌丝变粗;合欢枯萎病菌Fusarium oxysporum的菌丝变细;苹果霉心病菌Trichothecium roseum菌丝形态无明显变化。同时发现,不同病原菌菌丝色素产生的速度均受到了抑制。表明该枯草芽孢杆菌所产生的挥发性物质对不同病原菌的抑菌效果和 作用方式不同。 初步研究了分离自石榴果实中的一株枯草芽孢杆菌Bacillus subtilis所产生的挥发性物质对几种常见林果病原菌的抑菌作用。发现该挥发性物质对不同病原菌菌丝生长具有一定的抑制作用,其中对杨树溃疡病菌 Botryosphaeria dothidea的抑制率达60%。将不同病原菌株与该挥发性物质互作后的菌丝形态与正常菌丝进行比较,结果表明：处理后的杨树溃疡病菌、石榴疮痂病菌 B.dothidea、苹果轮纹病菌B.dothidea及葡萄白腐病菌Coniothyrium diplodiella的菌丝分支增多,扭曲,菌丝顶端和中部细胞膨大;杨树腐烂病菌Valsa sordida及桑紫纹羽病菌Helicobasidium purpureum的菌丝变粗;合欢枯萎病菌Fusarium oxysporum的菌丝变细;苹果霉心病菌Trichothecium roseum菌丝形态无明显变化。同时发现,不同病原菌菌丝色素产生的速度均受到了抑制。表明该枯草芽孢杆菌所产生的挥发性物质对不同病原菌的抑菌效果和 作用方式不同。 |
| [23] | . Soil fungistasis can adversely affect the germination and growth of most fungal species in the field. Among the inhibitors, volatiles of microbial origins are potentially very important. In this study, we investigated the frequency and identity of bacteria producing fungistatic volatiles. Among the 1018 bacterial isolates tested, 328 were found to produce antifungal volatiles that could inhibit spore germination and mycelial growth of two nematicidal fungi Paecilomyces lilacinus and Pochonia chlamydosporia. A phylogenetic analysis based on restriction fragment length polymorphism (RFLP) and 16S rDNA sequence placed the 328 bacteria in five groups: Alcaligenaceae, Bacillales, Micrococcaceae, Rhizobiaceae and Xanthomonadaceae. Volatile compounds of 39 bacterial isolates were identified by gas chromatography/mass spectrum (GC/MS). Tests with commercially available antifungal compounds suggested that seven volatile compounds of bacterial origins (acetamide, benzaldehyde, benzothiazole, 1-butanamine, methanamine, phenylacetaldehyde and 1-decene) likely play important roles in soil fungistasis. |
| [24] | . |
| [25] | . Bacteria, isolated from canola and soybean plants, produced antifungal organic volatile compounds. These compounds inhibited sclerotia and ascospore germination, and mycelial growth of Sclerotinia sclerotiorum, in vitro and in soil tests. Ascospore germination in cavity slides was inhibited 54–90% by the volatile producers. When mycelial plugs or the sclerotia, exposed to these volatiles, were transferred to fresh agar plates, the pathogen could not grow, indicating the fungicidal nature of the volatiles. Head space volatiles, produced by bacteria, were trapped with activated charcoal, by passing nitrogen continuously over shake cultures for 48 h. The compounds were eluted from the charcoal with methylene chloride and identified using Gas Chromatography–Mass Spectrometry (GC–MS). The volatile compounds included aldehydes, alcohols, ketones and sulfides. Of the 23 compounds assayed for antifungal activity in divided Petri plates, with filter-disks soaked with these compounds (100 and 150 μl), only six compounds completely inhibited mycelial growth or sclerotia formation, suggesting their potential role in biological control. The compounds are benzothiazole, cyclohexanol, n-decanal, dimethyl trisulfide, 2-ethyl 1-hexanol, and nonanal. Volatiles may play an important role in the inhibition of sclerotial activity, limiting ascospore production, and reducing disease levels. Studies are under way to understand this phenomenon under field conditions. This is the first report on the identification and use of bacterial antifungal organic volatiles in biocontrol. |
| [26] | . Microbial interactions via infochemicals are fundamental to the development of spatial distribution and activity variations in ecosystems. Microorganisms produce a wide range of infochemicals, frequently secondary metabolites, most of which are soluble and many volatile. Volatile organic compounds (VOCs) have been identified in soil atmospheres and related to community structure and function. VOC profiles produced by microorganisms are consistent, relating to cultural conditions, environment and inputs, and so to population and function dynamics. VOC-mediated interactions can result in functional responses by the organisms involved that result in selective advantage to some community members. Positive, negative or neutral interactions can occur between a very wide range of soil bacteria and fungi. These effects include both stimulation and inhibition of growth, by 40 and 60%, respectively, and enzyme production. These effects are usually transient, e.g. removal of an antagonist is followed by complete recovery. Up- and down-regulation of gene expression, by mRNA and protein profiling has been demonstrated. VOCs have played an important role during the evolution of microorganisms in the context of their communities. |
| [27] | |
| [28] | |
| [29] | The antifungal effects of volatile compounds produced by Bacillus strains; Bacillus subtilis PPCB001 or Bacillus amyloliquefaciens PPCB004 and antagonist combination (PPCB001 + PPCB004) against Penicillium digitatum Sacc., Penicillium italicum Wehmer and Penicillium crustosum Thom isolates were investigated in vitro and in vivo. The antagonists alone or in combination inhibited the radial mycelial growth of Penicillium spp. in vitro. Among the three Penicillium isolates tested P. crustosum showed 73.3% of mycelial growth inhibition in presence of PPCB004. The antifungal effects of volatiles increased with increasing time (days), and PPCB004 showed the highest inhibition of radial mycelial growth in P. crustosum on the 10th day. The EC 50 was 2.5 × 10 5 CFU ml 611 for PPCB001; 9.45 × 10 6 CFU ml 611 for PPCB004 and 7.76 × 10 6 CFU ml 611 for PPCB001 + PPCB004. Antagonist PPCB004 incubated at 37 °C for 24 h showed higher inhibitory effect on spore germination and germ tube elongation in P. crustosum than all other treatments. Although, PPCB001 produced a higher number of volatile compounds (21 different types) than PPC004 (8 different types), 3-hydroxy-2-butanone (acetoin) was the predominant ketone produced by both PPCB001 (45.98%) and PPCB004 (97.52%). Antagonist PPCB004 showed significant inhibition on decay incidence and severity in Valencia, inoculated with P. crustosum and held at 25 °C for 12 days. The observations indicated that B. amyloliquefaciens PPCB004 to control P. crustosum in postharvest systems. |
| [30] | . 通过检索与总结近年来国内外有关生姜挥发油提取方法、化学成分和药理活性的相关文献,为生姜挥发油的研究、开发与运用提供参考依据.生姜挥发油的提取方法主要包括水蒸汽蒸馏法、压榨法和超临界CO2萃取法,化学成分主要归属单萜烯类、单萜烯类氧化物、倍半萜烯类、倍半萜烯类氧化物4大类,具有明显的抗氧化、抑菌、镇痛、保肝、中枢调节,及缺血性脑损伤神经保护作用等药理活性,在医药、食品、香料和化妆品等领域具有广阔的应用前景. . 通过检索与总结近年来国内外有关生姜挥发油提取方法、化学成分和药理活性的相关文献,为生姜挥发油的研究、开发与运用提供参考依据.生姜挥发油的提取方法主要包括水蒸汽蒸馏法、压榨法和超临界CO2萃取法,化学成分主要归属单萜烯类、单萜烯类氧化物、倍半萜烯类、倍半萜烯类氧化物4大类,具有明显的抗氧化、抑菌、镇痛、保肝、中枢调节,及缺血性脑损伤神经保护作用等药理活性,在医药、食品、香料和化妆品等领域具有广阔的应用前景. |
| [31] | . 对人参茎叶挥发油的有效成分进行分离纯化，利用薄层层析（TLC）、红外光谱（IR）、气相色谱（GC）、气相色谱－质谱（GC／MS）联机技术从该挥发油中分离鉴定6个倍半萜烯类化合物。 . 对人参茎叶挥发油的有效成分进行分离纯化，利用薄层层析（TLC）、红外光谱（IR）、气相色谱（GC）、气相色谱－质谱（GC／MS）联机技术从该挥发油中分离鉴定6个倍半萜烯类化合物。 |
| [32] | The chemical composition of the aerial parts essential oils of Eryngium caucasicum Trautv. was studied by a combination of GC and GC-MS. Studies were carried out on leaves and stem oils obtained from coastal and hill slope areas at vegetative and generative phases. Fifty five components of the oil were identified representing 100 % of the oil. The main constituents of the oil were 3-hexyne (46.1%), 0205-sesquiphellandrene (20.4 %) and limonene (10.7 %) at first vegetative phase (May, 2009) in the leaves of coastal plants, while 5-methyl-2-pyrimidone (53.4 %), limonene (12.8 %) and 6-acetoxy-2,3-dihydro-1H-pyrrolizin (12.4 %) were dominant components in hill slope. The major components of the essential oils at second vegetative phase (Jun, 2009) in the leaves of coastal plants were 4 (5)-acetyl-1H-imidazole (63.6 %), thymol (13.9 %) and 0205-sesquiphellandrene (10.0 %), and in hill slope plants were 0205-sesquiphellandrene (44.3 %), limonene (20.1 %) and trans-0205-farnesene (14.1). 5-Methyl-2-pyrimidone (74.9 %), 4-(1,5-dimethylhex-4-enyl)cyclohex-2-enone (15.8 %) and 0205-sesquiphellandrene (2.9 %) were dominant components at generative phase (Jul, 2009) in the stems of coastal plants, while 0205-sesquiphellandrene (25.8 %), 5-methyl-2-pyrimidone (18.7 %) and limonene (11.8 %) were dominant components in hill slope plants. |
| [33] | . . |
| [34] | . Natural Products As Sources of New Drugs over the 30 Years from 1981 to 2010 |
| [35] | |
| [36] | In plants, sesquiterpenes of different structural types are biosynthesized from the isoprenoid intermediate farnesyl diphosphate. The initial reaction of the biosynthesis is catalyzed by sesquiterpene cyclases (synthases). In Artemisia annua L. (annual wormwood), a number of such sesquiterpene cyclases are active. We have isolated a cDNA clone encoding one of these, amorpha-4,11-diene synthase, a putative key enzyme of artemisinin biosynthesis. This clone contains a 1641-bp open reading frame coding for 546 amino acids (63.9 kDa), a 12-bp 5′-untranslated end, and a 427-bp 3′-untranslated sequence. The deduced amino acid sequence is 32 to 51% identical with the sequence of other known sesquiterpene cyclases from angiosperms. When expressed in Escherichia coli, the recombinant enzyme catalyzed the formation of both olefinic (97.5%) and oxygenated (2.5%) sesquiterpenes from farnesyl diphosphate. GC–MS analysis identified the olefins as (E)-β-farnesene (0.8%), amorpha-4,11-diene (91.2%), amorpha-4,7(11)-diene (3.7%), γ-humulene (1.0%), β-sesquiphellandrene (0.5%), and an unknown olefin (0.2%) and the oxygenated sesquiterpenes as amorpha-4-en-11-ol (0.2%) (tentatively), amorpha-4-en-7-ol (2.1%), and α-bisabolol (0.3%) (tentatively). Using geranyl diphosphate as substrate, amorpha-4,11-diene synthase did not produce any monoterpenes. The recombinant enzyme has a broad pH optimum between 7.5 and 9.0 and the Km values for farnesyl diphosphate, Mg2+, and Mn2+ are 0.9, 70, and 13 μM, respectively, at pH 7.5. A putative reaction mechanism for amorpha-4,11-diene synthase is suggested. |
| [37] | |
| [38] | Considering that as many as 80% of the anticancer drugs have their roots in natural products derived from traditional medicine, we examined compounds other than curcumin from turmeric (Curcuma longa) that could exhibit anticancer potential. Present study describes the isolation and characterization of another turmeric-derived compound, β-sesquiphellandrene (SQP) that exhibits anticancer potential comparable to that of curcumin. We isolated several compounds from turmeric, including SQP, α-curcumene, ar-turmerone, α-turmerone, β-turmerone, and γ-turmerone, only SQP was found to have antiproliferative effects comparable to those of curcumin in human leukemia, multiple myeloma, and colorectal cancer cells. While lack of the NF-κB-p65 protein had no effect on the activity of SQP, lung cancer cells that expressed p53 were more susceptible to the cytotoxic effect of SQP than were cells that lacked p53 expression. SQP was also found to be highly effective in suppressing cancer cell colony formation and inducing apoptosis, as shown by assays of intracellular esterase activity, plasma membrane integrity, and cell-cycle phase. SQP was found to induce cytochrome c release and activate caspases that lead to poly ADP ribose polymerase cleavage. SQP exposure was associated with downregulation of cell survival proteins such cFLIP, Bcl-xL, Bcl-2, c-IAP1, and survivin. Furthermore, SQP was found to be synergistic with the chemotherapeutic agents velcade, thalidomide and capecitabine. Overall, our results indicate that SQP has anticancer potential comparable to that of curcumin. |
| [39] | |
| [40] | OBPs have been recently demonstrated to be required for odour perception in insects and directly involved in odour discrimination. In aphids they might represent new interesting targets for the control of their population in agriculture. Based on sequence information available in the EST database, we have cloned four genes encoding odorant-binding proteins (OBP) in Acyrthosiphon pisum and homologous genes in other aphid species. Unlike OBPs from other orders of insects, that are greatly divergent, in aphids these proteins have been found to be highly conserved, with differences between species limited to only few amino acid substitutions. On the contrary, similarities between OBP sequences of the same species are poor with 31% or less of identical amino acids. Three selected OBPs (OBP1, OBP3 and OBP8) have been expressed in bacteria and purified. Ligand-binding experiments have shown similar behaviour of the three proteins towards several organic compounds, but also some significant selectivities. In particular, ( E)-尾-farnesene, the alarm pheromone and its related compound farnesol exhibited good affinity to OBP3, but did not bind the other two proteins. We suggest that OBP3 could mediate response of aphids to the alarm pheromone. |
| [41] | |
| [42] | . In order to make clear the diurnal variation of plant volatiles，we collected the VOCs from Cinnamomum camphora flowers using the dynamic headspace air-circulation method at 7：30，10：00，12：30，15：00 and 17：30，identified the main compounds of VOCs with thermal desorption system/gas chromatography/mass spectrum （TDS-GC-MS）. Results showed that C. camphora flowers emitted 51 types of VOCs，mainly as terpenoids. At 15：00，species and emission of the VOCs were highest （40 species and 8 049.5 peak area units），mainly as linalool （relative content 37.0%），epoxylinalol （12.7%）， （Z-ocimene （7.2%） and perillene（6.2%）. Thus，increases in species and emissions of the VOCs from C. camphora flowers at 15：00 are both visibly increased revealed a possible affinity have strong relevance for strong light and high temperature stress. ［Ch，3 fig. 1 tab. 15 ref.］ In order to make clear the diurnal variation of plant volatiles，we collected the VOCs from Cinnamomum camphora flowers using the dynamic headspace air-circulation method at 7：30，10：00，12：30，15：00 and 17：30，identified the main compounds of VOCs with thermal desorption system/gas chromatography/mass spectrum （TDS-GC-MS）. Results showed that C. camphora flowers emitted 51 types of VOCs，mainly as terpenoids. At 15：00，species and emission of the VOCs were highest （40 species and 8 049.5 peak area units），mainly as linalool （relative content 37.0%），epoxylinalol （12.7%）， （Z-ocimene （7.2%） and perillene（6.2%）. Thus，increases in species and emissions of the VOCs from C. camphora flowers at 15：00 are both visibly increased revealed a possible affinity have strong relevance for strong light and high temperature stress. ［Ch，3 fig. 1 tab. 15 ref.］ |
| [43] | . . |
