桑树内生拮抗细菌Burkholderia cepacia Lu10-1的分离鉴定及其侵染定殖规律研究
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摘要
桑树(Morus alba L.)在我国的栽培已有七千多年的历史,是蚕业生产的重要物质基础,也是传统的药用植物。但近年来桑树病害的发生表现出逐年上升的趋势,严重阻碍了蚕桑生产的可持续发展。现阶段对各种桑树病害的防治很大程度上依赖于化学农药的使用,由此带来的环境污染、农药残留等诸多问题引起人们广泛关注。因而,寻求对桑树病害合理有效的防治措施已势在必行。
作为一类新型的生防因子,植物内生细菌是定殖在健康植物的各种组织和器官中,与植物之间形成互惠共存、相互制约的和谐联合关系的一类微生物。其不易受环境条件的影响,可在植物体内长期定殖和传导,更有利于发挥生防作用,已成为植物病害生物防治上一类极具应用潜能的资源菌。因此,从桑树体内分离筛选有益细菌应用于桑树病害的生物防治具有重要的现实意义,但目前尚未见有桑树内生细菌的相关研究报道。
鉴于此,本研究对从健康桑树叶片中分离到的一株内生拮抗细菌Lu10-1进行了系统的分类学鉴定,并测定了该内生细菌在桑树体内的定殖数量消长动态,探讨了该菌株在桑树体内的侵染定殖规律。主要研究结果如下:
1对桑树内生拮抗细菌Lu10-1进行了抑菌谱的测定。结果表明,菌株Lu10-1对9株供试植物病原真菌、2株革兰氏阴性及1株革兰氏阳性病原细菌均表现出较强的抗菌活性,抑菌谱较广;病原菌中包括桑炭疽病菌(Colletotrichum morifolium)、桑粘格孢菌(Septogloeum mori)、桑丁香假单孢杆菌(Pseudomonas syringae pv. mori)三株桑树主要病原菌。
2对菌株Lu10-1进行了系统的分类学鉴定。结果表明,Lu10-1菌株归属于洋葱伯克霍尔德氏菌基因型Ⅰ(Burkholderia cepacia genomovarⅠ),菌株的16S rDNA序列已在GenBank中注册,登录号为EF546394。
3采用双抗药性标记法测定了Burkholderia cepacia Lu10-1在桑树体内的数量消长动态。结果表明,Lu10-1菌株可通过浸种、浸根、涂叶和针刺等接种方法进入桑树体内定殖,并证实了该内生细菌能够在桑树体内不同组织之间转移和传导。Lu10-1菌株浸种接种后,细菌在桑树体内的定殖数量总体上呈现下降趋势,到第20天后菌量趋于稳定;细菌浸根接种后,在根、茎和叶中定殖的菌量均呈现出“先增后降”的趋势。该菌株可在桑树体内长期定殖,且在定殖过程中菌株的拮抗性能未改变。
4用扫描电子显微镜观察了Burkholderia cepacia Lu10-1对桑树根的吸附和侵染情况。结果表明,Lu10-1菌株在桑树根各区的吸附和分布存在较大差异,根成熟区根毛表面和伸长区为内生细菌Lu10-1在根表面吸附的优先位点。该菌可从主根与侧根的交联处、根毛分生处、根毛表面、外表皮裂痕以及根外表皮细胞的间隙等部位直接侵入到桑树根表皮内。
5用gfp基因标记了Burkholderia cepacia Lu10-1。结果表明,标记细菌在481 nm的蓝光激发下发出明亮的绿色荧光,gfp基因在Lu10-1菌株中得到了很好的表达。且pGFP4412质粒在菌株Lu10-1中的遗传稳定性较强,可以满足在桑树体内实时监测的需要。
6用激光共聚焦扫描显微镜实时监测了Burkholderia cepacia Lu10-1在桑树体内的侵染、转移、传导和定殖全过程。结果表明,该菌主要从主根与侧根交联处,根毛分生处和根毛进入桑树根的皮层和中柱,通过输导组织向桑树茎部、叶柄和叶片中迁移,主要定殖在桑树各维管组织和皮层的细胞间隙,细胞内未发现标记细菌的定殖。
Mulberry (Morus alba L.), an important substantial basis of sericultural industry, is widely planted about 7000 years’history in our country. In addition, it is also traditional officinal plant. However, it is always infected with many diseases in the past years, which has embarrassed severely the sustainable development of sericicultural industry. Presently pesticides application is the major messure of controlling the disease, but the use of pesticides is restricted because this may induce chemical residues in the environment. Compared with chemical control, biological control is more practical and safer in preventing the mulberry pathogens.
Endophytic bacterum, which colonize in the host plant tissues without doing substantive harm to the host or gaining benefit other than securing residency, have successfully served as biocontrol agents in recent years. In addition, endophytic bacteria can exit in the plant well, and control disease stably. Consequently, endophytic bacteria are good biocontrol bacteria to control mulberry disease. However, little report could be found focusing the endophytic bacteria of mulberry.
Lu10-1 strain, isolated from the healthy mulberry leaves, was an antagonistic bacterium that exhibited excellent resistant against various plant pathogen such as Ralstonia solanacearum and Colletotrichum morifolium hara. In this study, the final taxonomical status of strain Lu10-1 was confirmed to be a single species with the phenotypic and molecular methods for species identification. In addition, the population of strain Lu10-1 living in the mulberry tissues was analyzed, and the endophytic colonization rule of Lu10-1 was established with mulberry seedlings following different methods. The main results were showed as the followings:
1. The result showed that strain Lu10-1 had broad antimicrobial spectrum by screening its antagonistic activity against Colletotrichum morifolium, Septogloeum mori, Pseudomonas syringae pv. Mori, and so on.
2. Strain Lu10-1 was identified based on the analysis of its 16S rRNA gene sequence homology, the physiological and biochemical characteristics, and the recA gene sequence comparison. The results showed that Lu10-1 belonged to Burkholderia sp.. In the phylogenetic tree, Lu10-1 was the closest relative to Burkholderia cepacia (X80284) with more than 98% sequences similarity. The 16S rDNA sequences of Lu10-1 have been registered at GenBank database under the accession number EF546394. Finally, it was clearly demonstrated that the strain Lu10-1 fell into Burkholderia cepacia complex genomovarⅠby PCR with Bcc-specific and genomovar-specific primers based on recA gene. The final taxonomical status of strain Lu10-1 was confirmed to be a single species.
3. Tagged with the drug resistance of Rif. (300μg/mL) and Amp.(300μg/mL), the strain Lu10-1 was inoculated by the way of stem and leaf acupuncturing, seed soaking, root soaking and leaf daubing. The population of strain Lu10-1 living in the mulberry tissues decreased as a whole after the treatment of seed dipping and root dipping. As for the population of strain Lu10-1 in mulberry leaves and stems after the treatments, it increased first then decreaed. Ultimately, it achieved a steady level in different mulberry tissues after 20 days. In the investigation of the colonization, it was proved that the strain Lu10-1 could colonize and transmit in the mulberry, and its resistance against plant pathogen did not change in the process of colonization compared to the original strains. Therefore, Burkholderia cepacia Lu10-1 would have a far future in the biological control of mulberry disease.
4. The adsorption, multiplication and penetration of the Burkholderia cepacia Lu10-1 in the young roots of mulberry were examined with scanning electron microscopy at the different inoculation. The results showed as follows: The root hair surface and the region of elongation surface of mulberry were two preferential sites of adsorption and multiplication of the strain Lu10-1. The bacteria could enter the epidermal cells of mulberry roots by the lateral root junction, the root hair junction, the root hair surface, the cracks between epidermal cells, and epidermal cells of the young lateral root.
5. In this study, we had maked the Burkholderia cepacia Lu10-1 with gfp gene. The results showed that the vector pGFP4412 produced high level of GFP, and the transformants appeared bright green under blue light. The esperiment of stability of the gfp-labeled strain indicated that its stability is very strong. Therefore, the strain of Lu10-1-gfp could statisfy the need of study in the colonization in the interior of the mulberry tissues.
6. Microscopic observation of colonization of Burkholderia cepacia Lu10-1-gfp inside the mulberry tissues using confocal laser scanning microscope showed that the Burkholderia cepacia Lu10-1 could colonize and transmit in the mulberry. The bacterial cell infected mainly the inner cortex and vascular stele of mulberry root from the root hair, and the junction between primary and lateral root. By the vascular tissue, the bacteria migrated slowly from roots to stems and leaves. The bacteria were mainly located in the intercellular spaces and vascular tissue of mulberry. Otherwise, the strain Lu10-1 could not enter interior of the mulberry cells.
作为一类新型的生防因子,植物内生细菌是定殖在健康植物的各种组织和器官中,与植物之间形成互惠共存、相互制约的和谐联合关系的一类微生物。其不易受环境条件的影响,可在植物体内长期定殖和传导,更有利于发挥生防作用,已成为植物病害生物防治上一类极具应用潜能的资源菌。因此,从桑树体内分离筛选有益细菌应用于桑树病害的生物防治具有重要的现实意义,但目前尚未见有桑树内生细菌的相关研究报道。
鉴于此,本研究对从健康桑树叶片中分离到的一株内生拮抗细菌Lu10-1进行了系统的分类学鉴定,并测定了该内生细菌在桑树体内的定殖数量消长动态,探讨了该菌株在桑树体内的侵染定殖规律。主要研究结果如下:
1对桑树内生拮抗细菌Lu10-1进行了抑菌谱的测定。结果表明,菌株Lu10-1对9株供试植物病原真菌、2株革兰氏阴性及1株革兰氏阳性病原细菌均表现出较强的抗菌活性,抑菌谱较广;病原菌中包括桑炭疽病菌(Colletotrichum morifolium)、桑粘格孢菌(Septogloeum mori)、桑丁香假单孢杆菌(Pseudomonas syringae pv. mori)三株桑树主要病原菌。
2对菌株Lu10-1进行了系统的分类学鉴定。结果表明,Lu10-1菌株归属于洋葱伯克霍尔德氏菌基因型Ⅰ(Burkholderia cepacia genomovarⅠ),菌株的16S rDNA序列已在GenBank中注册,登录号为EF546394。
3采用双抗药性标记法测定了Burkholderia cepacia Lu10-1在桑树体内的数量消长动态。结果表明,Lu10-1菌株可通过浸种、浸根、涂叶和针刺等接种方法进入桑树体内定殖,并证实了该内生细菌能够在桑树体内不同组织之间转移和传导。Lu10-1菌株浸种接种后,细菌在桑树体内的定殖数量总体上呈现下降趋势,到第20天后菌量趋于稳定;细菌浸根接种后,在根、茎和叶中定殖的菌量均呈现出“先增后降”的趋势。该菌株可在桑树体内长期定殖,且在定殖过程中菌株的拮抗性能未改变。
4用扫描电子显微镜观察了Burkholderia cepacia Lu10-1对桑树根的吸附和侵染情况。结果表明,Lu10-1菌株在桑树根各区的吸附和分布存在较大差异,根成熟区根毛表面和伸长区为内生细菌Lu10-1在根表面吸附的优先位点。该菌可从主根与侧根的交联处、根毛分生处、根毛表面、外表皮裂痕以及根外表皮细胞的间隙等部位直接侵入到桑树根表皮内。
5用gfp基因标记了Burkholderia cepacia Lu10-1。结果表明,标记细菌在481 nm的蓝光激发下发出明亮的绿色荧光,gfp基因在Lu10-1菌株中得到了很好的表达。且pGFP4412质粒在菌株Lu10-1中的遗传稳定性较强,可以满足在桑树体内实时监测的需要。
6用激光共聚焦扫描显微镜实时监测了Burkholderia cepacia Lu10-1在桑树体内的侵染、转移、传导和定殖全过程。结果表明,该菌主要从主根与侧根交联处,根毛分生处和根毛进入桑树根的皮层和中柱,通过输导组织向桑树茎部、叶柄和叶片中迁移,主要定殖在桑树各维管组织和皮层的细胞间隙,细胞内未发现标记细菌的定殖。
Mulberry (Morus alba L.), an important substantial basis of sericultural industry, is widely planted about 7000 years’history in our country. In addition, it is also traditional officinal plant. However, it is always infected with many diseases in the past years, which has embarrassed severely the sustainable development of sericicultural industry. Presently pesticides application is the major messure of controlling the disease, but the use of pesticides is restricted because this may induce chemical residues in the environment. Compared with chemical control, biological control is more practical and safer in preventing the mulberry pathogens.
Endophytic bacterum, which colonize in the host plant tissues without doing substantive harm to the host or gaining benefit other than securing residency, have successfully served as biocontrol agents in recent years. In addition, endophytic bacteria can exit in the plant well, and control disease stably. Consequently, endophytic bacteria are good biocontrol bacteria to control mulberry disease. However, little report could be found focusing the endophytic bacteria of mulberry.
Lu10-1 strain, isolated from the healthy mulberry leaves, was an antagonistic bacterium that exhibited excellent resistant against various plant pathogen such as Ralstonia solanacearum and Colletotrichum morifolium hara. In this study, the final taxonomical status of strain Lu10-1 was confirmed to be a single species with the phenotypic and molecular methods for species identification. In addition, the population of strain Lu10-1 living in the mulberry tissues was analyzed, and the endophytic colonization rule of Lu10-1 was established with mulberry seedlings following different methods. The main results were showed as the followings:
1. The result showed that strain Lu10-1 had broad antimicrobial spectrum by screening its antagonistic activity against Colletotrichum morifolium, Septogloeum mori, Pseudomonas syringae pv. Mori, and so on.
2. Strain Lu10-1 was identified based on the analysis of its 16S rRNA gene sequence homology, the physiological and biochemical characteristics, and the recA gene sequence comparison. The results showed that Lu10-1 belonged to Burkholderia sp.. In the phylogenetic tree, Lu10-1 was the closest relative to Burkholderia cepacia (X80284) with more than 98% sequences similarity. The 16S rDNA sequences of Lu10-1 have been registered at GenBank database under the accession number EF546394. Finally, it was clearly demonstrated that the strain Lu10-1 fell into Burkholderia cepacia complex genomovarⅠby PCR with Bcc-specific and genomovar-specific primers based on recA gene. The final taxonomical status of strain Lu10-1 was confirmed to be a single species.
3. Tagged with the drug resistance of Rif. (300μg/mL) and Amp.(300μg/mL), the strain Lu10-1 was inoculated by the way of stem and leaf acupuncturing, seed soaking, root soaking and leaf daubing. The population of strain Lu10-1 living in the mulberry tissues decreased as a whole after the treatment of seed dipping and root dipping. As for the population of strain Lu10-1 in mulberry leaves and stems after the treatments, it increased first then decreaed. Ultimately, it achieved a steady level in different mulberry tissues after 20 days. In the investigation of the colonization, it was proved that the strain Lu10-1 could colonize and transmit in the mulberry, and its resistance against plant pathogen did not change in the process of colonization compared to the original strains. Therefore, Burkholderia cepacia Lu10-1 would have a far future in the biological control of mulberry disease.
4. The adsorption, multiplication and penetration of the Burkholderia cepacia Lu10-1 in the young roots of mulberry were examined with scanning electron microscopy at the different inoculation. The results showed as follows: The root hair surface and the region of elongation surface of mulberry were two preferential sites of adsorption and multiplication of the strain Lu10-1. The bacteria could enter the epidermal cells of mulberry roots by the lateral root junction, the root hair junction, the root hair surface, the cracks between epidermal cells, and epidermal cells of the young lateral root.
5. In this study, we had maked the Burkholderia cepacia Lu10-1 with gfp gene. The results showed that the vector pGFP4412 produced high level of GFP, and the transformants appeared bright green under blue light. The esperiment of stability of the gfp-labeled strain indicated that its stability is very strong. Therefore, the strain of Lu10-1-gfp could statisfy the need of study in the colonization in the interior of the mulberry tissues.
6. Microscopic observation of colonization of Burkholderia cepacia Lu10-1-gfp inside the mulberry tissues using confocal laser scanning microscope showed that the Burkholderia cepacia Lu10-1 could colonize and transmit in the mulberry. The bacterial cell infected mainly the inner cortex and vascular stele of mulberry root from the root hair, and the junction between primary and lateral root. By the vascular tissue, the bacteria migrated slowly from roots to stems and leaves. The bacteria were mainly located in the intercellular spaces and vascular tissue of mulberry. Otherwise, the strain Lu10-1 could not enter interior of the mulberry cells.
引文
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