孜然提取物抑菌活性的研究
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摘要
本研究以小麦条锈病菌(Puccinia striformis)、稻瘟病菌(Pyricularia grisea)、黄瓜霜霉病菌(Pseudoperonispora cubensis)和水稻白叶枯病菌(Xanthomonas campestris pv. oryzae)等12种植物病原菌为供试菌种，采用离体活性与活体盆栽相结合的测定方法，主要对孜然抑菌活性作了较为系统的研究，初步得出如下结果：
1．在离体测定中，孜然提取物与其它植物提取物相比较，孜然粗提物抑菌活性表现最为突出，对小麦纹枯病菌、苹果霉心病菌、白菜黑斑病菌、棉花枯萎病菌、杨树溃疡病菌等5种病原菌的菌丝生长抑制率均为100％，对稻瘟病菌和黄瓜炭疽病菌的抑制率分别为89.5％、78.7％。经毒力测定，孜然粗提物对小麦纹枯病菌的毒力最好，EC_(50)为3.6563g/L。在孢子萌发试验中，孜然粗提物对稻瘟病菌、小麦光腥黑穗病菌的孢子萌发抑制率均在80％以上，说明孜然提取物的抑菌范围是非常广泛的。
2．病原菌形态观察结果表明苦豆子粗提物能使黄瓜炭疽病菌的菌丝产生许多畸形的膨大瘤状节，孜然及其它植物样品粗提物对供试病原真菌的菌丝均无明显异常表现。
3．在盆栽试验中，孜然与其它11种植物相比较，除苍耳粗提物对病害的防治表现不错外，孜然粗提物对病害的防治谱均比其它植物粗提物更广泛。孜然粗提物对稻瘟病、黄瓜炭疽病的保护和治疗作用均大于60％，对白菜黑斑病、黄瓜霜霉病的保护作用大于60％；经室内水培法试验，孜然粗提物对杨树溃疡病的治疗作用为60％。这说明孜然粗提物对真菌类的叶斑病害防效较好，对枝干溃疡性病害也具有一定的防治作用。从病原菌分类地位看，孜然粗提物的防病范围适用于鞭毛菌、子囊菌、半知菌。但试验中也发现孜然粗提物对小麦条锈病、小麦纹枯病的防效较差，对稻白叶枯细菌性病害无效。此外，通过试验可以看出，植物样品粗提物离体活性和活体活性之间不是完全有相关性的。
4．采用液-液分配萃取、薄层层析、逐级柱层析及生物活性测定相结合的跟踪测定方法，对孜然抑菌成分进行了分离。结果表明，孜然乙醇提取物的石油醚萃取物抑菌活性最高，对稻瘟菌等4种病菌菌丝生长抑制率及对黄瓜霜霉病、小麦白粉病的防效均在60％以上；石油醚萃取物经柱层析后得7部分，其中石07中有白色结晶，石06抑菌活性较高，对稻瘟菌等3种病菌菌丝生长抑制率及对小麦白粉病的防效均在60％以上；石06经柱层析后得6部分，其中F04抑菌活性较高，对稻瘟菌等3种病菌菌丝生长抑制率均在60％以上，对小麦白粉病的治疗作用达91.5％；F04经柱层析后得3部分，其中c03抑菌活性较高，对棉花枯萎病菌菌丝生长抑制率在60％以上；c03柱层析后得一活性成分单体ZYG，经结构鉴定为对异丙基苯甲酸，其具有较高的抑菌活性，当浓度为50 mg/L时，ZYG能完全抑制稻瘟病菌的菌丝生长，对杨树溃
疡病菌的菌丝生长抑制率为70%以上。
5.孜然提取物对小麦白粉病菌菌落线性长度增长的影响说明:接种72h、96h、
120h时，处理叶片上的菌落数比对照少，菌落线性平均长度比对照短，说明孜然提
取物能抑制小麦白粉病菌菌落扩展，进而降低了病原菌的繁殖能力。
Tested with Puccinia striiformis, Pyricularia grisea, Pseudoperonispora cubensi and Xanthomonas campestris pv.oryzae et al 12 kinds of fungi, the bioactivity of Cuminum cyminum was studied by a systematic way in vitro and pot test. The results are as follows:
Firstly, contested with the extracts of other 11 kinds of plants, the extract of Cuminum cyminum had the best bioactivity, it can completely inhibit the hypha growth of Rhizoctonia cerealis, Fusarium oxysporum f.sp. vasinfectum, Dothiorella gregaria, Trichoticum roseum, Alternaria okracea under the concentration of 0.05g/mL. The extract of Cuminum cyminum had the best inhibiting rate to the hypha growth of Rhizoctonia cerealis(EC50=3.6563 g/L). Furthermore, the extract had more than 80% inhibiting rates to the spore germination of Pyricularia grisea and Tilletia foetida under the concentration of 0.1g/mL.
Secondly, the observation of morphological character shows that the hypha of Colletotrichum lagenarium was deformed by the extract ofSophora alopecuroides.
Thirdly, the results of pot test indicated that the extract of Cuminum cyminum had more than 60% protective efficacy to the Black rot of Chinese cabbage, Rice blast, Cucumber downy mildew, Cucumber anthracnose and had more than 60% therapeutic efficacy to the Rice blast, Cucumber anthracnose and Poplar canker under the concentration of 0.04g/mL. Furthermore, the results shows that there isn't good correlation between efficacy in vitro and efficacy in vivo.
Fourthly, with the help of bioassay, an ingredient (ZYG) and a white crystal were got from Cuminum cyminum. The molecular structure of ZYG was found to be p-isopropyl benzoic acid, the ZYG can inhibit the hypha growth of Pyricularia grisea completely and had more than 70% inhibiting rates to the hypha growth of Dothiorella gregaria under the concentration of 50mg/L.
Finally, tested with the extract of Cuminum cyminum, the histopathological manifestation of Wheat powdery mildew was examined by acetic acid-ethyl alcohol for fixing-transparency and cotton-blue for staining. The results show that the colony expanding was suppressed.
1．在离体测定中，孜然提取物与其它植物提取物相比较，孜然粗提物抑菌活性表现最为突出，对小麦纹枯病菌、苹果霉心病菌、白菜黑斑病菌、棉花枯萎病菌、杨树溃疡病菌等5种病原菌的菌丝生长抑制率均为100％，对稻瘟病菌和黄瓜炭疽病菌的抑制率分别为89.5％、78.7％。经毒力测定，孜然粗提物对小麦纹枯病菌的毒力最好，EC_(50)为3.6563g/L。在孢子萌发试验中，孜然粗提物对稻瘟病菌、小麦光腥黑穗病菌的孢子萌发抑制率均在80％以上，说明孜然提取物的抑菌范围是非常广泛的。
2．病原菌形态观察结果表明苦豆子粗提物能使黄瓜炭疽病菌的菌丝产生许多畸形的膨大瘤状节，孜然及其它植物样品粗提物对供试病原真菌的菌丝均无明显异常表现。
3．在盆栽试验中，孜然与其它11种植物相比较，除苍耳粗提物对病害的防治表现不错外，孜然粗提物对病害的防治谱均比其它植物粗提物更广泛。孜然粗提物对稻瘟病、黄瓜炭疽病的保护和治疗作用均大于60％，对白菜黑斑病、黄瓜霜霉病的保护作用大于60％；经室内水培法试验，孜然粗提物对杨树溃疡病的治疗作用为60％。这说明孜然粗提物对真菌类的叶斑病害防效较好，对枝干溃疡性病害也具有一定的防治作用。从病原菌分类地位看，孜然粗提物的防病范围适用于鞭毛菌、子囊菌、半知菌。但试验中也发现孜然粗提物对小麦条锈病、小麦纹枯病的防效较差，对稻白叶枯细菌性病害无效。此外，通过试验可以看出，植物样品粗提物离体活性和活体活性之间不是完全有相关性的。
4．采用液-液分配萃取、薄层层析、逐级柱层析及生物活性测定相结合的跟踪测定方法，对孜然抑菌成分进行了分离。结果表明，孜然乙醇提取物的石油醚萃取物抑菌活性最高，对稻瘟菌等4种病菌菌丝生长抑制率及对黄瓜霜霉病、小麦白粉病的防效均在60％以上；石油醚萃取物经柱层析后得7部分，其中石07中有白色结晶，石06抑菌活性较高，对稻瘟菌等3种病菌菌丝生长抑制率及对小麦白粉病的防效均在60％以上；石06经柱层析后得6部分，其中F04抑菌活性较高，对稻瘟菌等3种病菌菌丝生长抑制率均在60％以上，对小麦白粉病的治疗作用达91.5％；F04经柱层析后得3部分，其中c03抑菌活性较高，对棉花枯萎病菌菌丝生长抑制率在60％以上；c03柱层析后得一活性成分单体ZYG，经结构鉴定为对异丙基苯甲酸，其具有较高的抑菌活性，当浓度为50 mg/L时，ZYG能完全抑制稻瘟病菌的菌丝生长，对杨树溃
疡病菌的菌丝生长抑制率为70%以上。
5.孜然提取物对小麦白粉病菌菌落线性长度增长的影响说明:接种72h、96h、
120h时，处理叶片上的菌落数比对照少，菌落线性平均长度比对照短，说明孜然提
取物能抑制小麦白粉病菌菌落扩展，进而降低了病原菌的繁殖能力。
Tested with Puccinia striiformis, Pyricularia grisea, Pseudoperonispora cubensi and Xanthomonas campestris pv.oryzae et al 12 kinds of fungi, the bioactivity of Cuminum cyminum was studied by a systematic way in vitro and pot test. The results are as follows:
Firstly, contested with the extracts of other 11 kinds of plants, the extract of Cuminum cyminum had the best bioactivity, it can completely inhibit the hypha growth of Rhizoctonia cerealis, Fusarium oxysporum f.sp. vasinfectum, Dothiorella gregaria, Trichoticum roseum, Alternaria okracea under the concentration of 0.05g/mL. The extract of Cuminum cyminum had the best inhibiting rate to the hypha growth of Rhizoctonia cerealis(EC50=3.6563 g/L). Furthermore, the extract had more than 80% inhibiting rates to the spore germination of Pyricularia grisea and Tilletia foetida under the concentration of 0.1g/mL.
Secondly, the observation of morphological character shows that the hypha of Colletotrichum lagenarium was deformed by the extract ofSophora alopecuroides.
Thirdly, the results of pot test indicated that the extract of Cuminum cyminum had more than 60% protective efficacy to the Black rot of Chinese cabbage, Rice blast, Cucumber downy mildew, Cucumber anthracnose and had more than 60% therapeutic efficacy to the Rice blast, Cucumber anthracnose and Poplar canker under the concentration of 0.04g/mL. Furthermore, the results shows that there isn't good correlation between efficacy in vitro and efficacy in vivo.
Fourthly, with the help of bioassay, an ingredient (ZYG) and a white crystal were got from Cuminum cyminum. The molecular structure of ZYG was found to be p-isopropyl benzoic acid, the ZYG can inhibit the hypha growth of Pyricularia grisea completely and had more than 70% inhibiting rates to the hypha growth of Dothiorella gregaria under the concentration of 50mg/L.
Finally, tested with the extract of Cuminum cyminum, the histopathological manifestation of Wheat powdery mildew was examined by acetic acid-ethyl alcohol for fixing-transparency and cotton-blue for staining. The results show that the colony expanding was suppressed.
引文
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