【作者】 李其利；

【导师】 黄俊斌；

【作者基本信息】 华中农业大学 ， 植物病理学， 2011， 博士

【摘要】 链霉菌一类具有广泛实际用途和巨大经济价值的微生物类群,它广泛的存在于不同的自然生态环境中,种类繁多,代谢功能各异。在目前发现的数万种微生物来源的活性物质中,有近50%的都是来自于链霉菌。链霉菌可以产生种类繁多的农用抗生素,而且大多数已在农业生产中广泛应用,可防治真菌、细菌、线虫病害以及一些重要的害虫,有的还具有除草活性。本试验室在2007年7月发现了一株对稻瘟病菌有很强拮抗作用的链霉菌菌株JK-1,通过形态学,生理生化鉴定以及16SrDNA序列分析,将其鉴定为球孢链霉菌(Streptomyces golobisporus)。在此基础之上进一步对球孢链霉菌JK-1的防病潜能和防病机制进行了系统研究。主要研究结果如下：1.采用传统的形态学观察、生理生化测定以及16S rDNA分析,将菌株JK-1鉴定为球孢链霉菌(Streptomyces golobisporus)。2.研究了不同培养条件对链霉菌JK-1生长及其产生抗菌物质的影响。结果表明,在供试的7种培养基中,LB最有利于菌体生长,PSB最有利于产生抗菌物质。该菌在10-35℃范围内均可以生长并产生抗菌物质,25℃为菌体生长和产生抗菌物质的最适温度。链霉菌JK-1在PSB中培养6d菌体干重和抑菌活性均可达到最大值；适宜于JK-1生长及其产生抗菌物质的pH值范围为5-11,最适为pH6-8。该菌能够有效的利用多种碳源和氮源进行生长和产生抗菌物质。其中分别以甘油为碳源和酪氨酸为氮源时生长最好；以麦芽糖为碳源和硫酸铵为氮源时产生抗菌物质最多。3.研究了链霉菌JK-1产生的抗菌物质的稳定性。结果表明,链霉菌JK-1产生的抗菌物质在高温下和蛋白酶K处理的情况下活性显著降低,在酸性条件比较稳定,而pH≥12的碱性条件容易失活。硫酸铵可以将发酵液中的抗菌物质沉淀下来,超滤发现其分子量大于10 KD。4.研究了球孢链霉菌JK-1对稻瘟病菌的抑制作用及其防病效果,发现球孢链霉菌的发酵液能够抑制稻瘟菌菌丝生长、孢子萌发以及附着胞形成。分别将链霉菌JK-1的菌体悬浮液、发酵液、发酵滤液与稻瘟菌分生孢子液混合接种,发现浓度为108 CFU/mL的菌体悬浮液、浓度为10%的发酵液和发酵液滤液均能完全抑制稻瘟病的发生。温室盆栽试验结果表明,在喷雾接种稻瘟菌前12h和接种后2 h喷雾发酵滤液,其防病效果分别为89.1%和88.3%。利用组织学和扫描电镜观察了球孢链霉菌发酵滤液对稻瘟病菌在水稻叶片上侵染过程的影响,结果表明,在接种后3h前喷雾JK-1发酵滤液能抑制稻瘟病菌分生孢子的萌发以及附着胞的形成,从而控制稻瘟病的发生。5.研究了链霉菌JK-1产生的挥发性物质对番茄灰霉病的防病效果及其作用机制,结果表明挥发性物质可以抑制灰霉病菌菌丝生长、孢子萌发以及孢子形成,且随JK-1麦粒培养物量的增加其抑菌活性逐渐增强。在接种后不同时间对番茄进行熏蒸处理,发现接种后立即用120 g/L的链霉菌麦粒培养物熏蒸可以完全抑制灰霉病的发生。扫描电镜结果表明,挥发性物质能够抑制灰霉菌分生孢子在番茄果实上的萌发以及侵染菌丝形成,并且导致分生孢子和侵染菌丝畸形,从而达到防治灰霉病的目的。利用二乙酸荧光素(FDA)和碘化丙啶(PI)染色结果表明,经过30 g/L,60 g/L和120 g/L的球孢链霉菌麦粒培养物熏蒸处理6d后分别有59.4%,76.7%和85.3%的分生孢子不能存活。透射电镜观察结果表明,经过挥发性物质处理的分生孢子和菌丝细胞内的液泡数量明显增多,部分孢子细胞壁增厚,菌丝细胞出现质壁分离,细胞的完整性被破坏。6.研究了链霉菌JK-1产生的挥发性物质对柑橘青霉菌的抑制作用及其防病效果。结果表明,挥发性物质可以抑制柑橘青霉菌菌丝生长、孢子萌发以及孢子形成,并且可有效控制柑橘青霉病的发生。采用气相色谱/质谱联用(GC/MS)分析,从链霉菌麦粒培养物产生的挥发性物质中鉴定出41种挥发性有机化合物,这些物质分别属于烃类、酯类、醇类、酮类、醚类、有机酸等。从中选择了8种特殊的化合物进行了抑菌活性及防病效果测定,结果发现二甲基二硫醚、二甲基三硫醚和苯乙酮能抑制柑橘青霉菌菌丝生长、分生孢子的萌发及形成,可有效的防治柑桔青霉病。更多还原

【Abstract】 Streptomyces are among the most widely distributed group of microorganisms with extensive practical use and enormous economic value in nature. About 50% antibiotics are the secondary metabolites of Streptomyces in ten thousands activity substances that have been found in microbiology. Streptomyces have been evaluated as biocontrol agents against various plant pathogens since they produce an array of secondary metabolites such as enzyme inhibitors, herbicides and large number of antibiotics. In July 2007, a small contaminant colony appeared in a PDA plate of Magnaporthe grisea in a plant pathology laboratory at Huazhong Agricultural University, and it showed strong inhibition of the fungus. The colony was transferred onto a fresh plate, grown for identification using morphological and molecular methods, and named Streptomyces globisporus JK-1. And then, biocontrol potential and mode of action of S. globisporus JK-1 were also studied in the present paper. The main results are listed as follows:1. Based on morphological, cultural, biochemical and physiological characteristics, strain JK-1 was identified as Streptomyces globisporus JK-1.2. The effect of medium components (i.e. carbon and nitrogen sources) and other culture conditions on production of antifungal substances of S. globisporus JK-1 was investigated. The results showed that among the seven tested medium, the best medium for growth and antifungal substances production were LB and potato sucrose broth (PSB), respectively; for the growth of S. globisporus JK-1 on medium PSB, the temperature range was 10 to 35℃, with optimum of 25℃; the dry weight and the antifungal substances production reached the highest level after incubation at 25℃for 6 days; the favorable pH value for growth and antifungal substance production was 5-11, with the optimum of pH 6-8. Among the 12 tested carbon and nitrogen sources in basal mineral salts medium, glycerol and tyrosine were the most favorable for its growth; maltose and ammonium sulfate were the most favorable for its antifungal substances production.3. To determine the property of the antifungal substances, the culture filtrates and the crude antifungal substances were treated in various ways. Antifungal activity was stable after incubation at 120℃for 30 min or treated with Proteinase K at 37℃for 60 min. The antifungal activity of the antifungal substances was stable at acid conditions while inactive in strong alkalinity (pH≥12). No antifungal activity was tested in the supernate of the culture filtrates, and the most antifungal activity was tested in the precipitate after adding the ammonium sulfate. The results of ultrafiltration of crude antifungal substances showed that the molecular weight was above 10 KD.4. Culture filtrates of JK-1 inhibited mycelial growth of M. oryzae, and histological investigations showed that conidial germination and appressorial formation of M. oryzae were suppressed on detached rice leaves. When applied at 15μl per 5-cm-long detached leaf, washed cells of JK-1 at 108 CFU/ml could suppress disease incidence of rice blast caused by M. oryzae co-inoculated at 5×105 spores/ml, but disease incidence was not reduced when washed cells of JK-1 were used at 106 or 107 CFU/ml. Applying the culture filtrate on rice seedlings in the greenhouse at 2 hours post inoculation (HPI) with M. oryzae showed 88.3% disease reduction of rice blast, while culture filtrate application before pathogen inoculation showed even higher rates of disease reduction. Suppression of the infection process of M. oryzae on detached rice leaves was observed by light and scanning electron microscopy, showing inhibited conidial germination and reduced appressorial formation on rice leaves sprayed with culture filtrate before 3 HPI. Application of JK-1 as undiluted culture broth or filtrates to plant tissues did not cause any noticeable negative effects. These results indicate that the antifungal substance(s) in the culture filtrate of S. globisporus JK-1 can exhibit an inhibitory effect on M. oryzae and a suppressive effect on rice blast disease.5. Antifungal activity against Botrytis cinerea of volatile substances from S. globisporus JK-1 grown on autoclaved wheat seed was studied in vitro and in vivo. The volatiles suppressed mycelial growth of various plant pathogens in vitro, especially that of B. cinerea and Sclerotinia sclerotiorum. Conidial germination and sporulation of B. cinerea were suppressed in the presence of volatiles. Disease incidence and severity on fungal-inoculated tomato fruit (Lycopersicon esculentum) were inhibited when fumigated with 120 g/L wheat seed culture of S. globisporus JK-1. Suppression of the infection process of B. cinerea on tomato fruit was observed via scanning microscopy, showing inhibition of conidial germination and of infection hyphae formation on tomato fruit, as well as abnormal morphology of infection hyphae and conidia exposed to the volatiles. The viability of the conidia obtained from the volatile-treated and non-treated disease lesions was tested with the vital stains fluorescein diacetate (FDA) and propidium iodide (PI). Conidia fumigated with 30 g/L,60 g/L or 120 g/L wheat seed culture of S. globisporus JK-1 at 20℃for 6 days showed 59.4%,76.7%, or 85.3% reduction in viability, respectively. Transmission electron microscopy of fumigated and untreated B. cinerea showed excessive vesiculation or thickened cell walls in exposed conidia and increased vesiculation or strong retraction of plasma membrane in exposed hyphae. The results of present study provide a better understanding of the mode of action of volatiles from JK-1 on B. cinerea. The inhibition growth of B. cinerea both in vitro and in vivo studies showed that volatiles from S. globisporus JK-1 have potential for control of postharvest grey mold of tomato fruits through fumigant action.6. Antifungal activity against Penicillium italicum of volatile substances from S. globisporus JK-1 grown on autoclaved wheat seed was studied in vitro and in planta. Fungal spore germination and mycelial growth of P. italicum cultures as well as sporulation and disease incidence on fungal-inoculated fruit were suppressed in the presence of the volatiles. For naturally infected fruit, disease incidence was reduced from 25% to 7.5%. Suppression of the infection process of P. italicum on Shatang Mandarin fruits (Citrus microcarpa) was observed via scanning electronic microscopy, showing inhibited spore germination on the Shatang Mandarin, and abnormal morphology for conidiophores and hyphae exposed to the volatiles. Based on gas chromatography/mass spectrophotometric analyses,41 volatile organic compounds were identified from the volatiles of S. globisporus JK-1, and the most abundant compound was trans-1,10-dimethyl-trans-9-decalol (geosmin), an earthy smelling substance. Among these, technical grade formulations of eight were chosen for further study:phenylethyl alcohol, caryophyllene, dimethyl disulfide, dimethyl trisulfide, acetophenone, d-Iimonene, isoledene, and aromadendrene. D-limonene, isoledene and aromadendrene showed no observable antifungal activity in vitro and in planta at tested concentrations. Both phenylethyl alcohol and caryophyllene showed weak inhibitory activity in vitro but no significant efficacy against P. italicum on Shatang Mandarin. Dimethyl disulfide or dimethyl trisulfide showed antifungal activity in vitro and efficacious control on Shatang Mandarin at a concentration of 100μL per liter of airspace in treatment containers. Acetophenone showed antifungal activity in vitro at a concentration of 100μL L-1 and efficacious control on Shatang Mandarin at the highest concentration of 1000μL L-1. Volatiles from S. globisporus JK-1 have potential for control of blue mold of citrus species through fumigant action.更多还原