HLB感染柑桔韧皮部内生细菌区系的研究
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摘要
柑橘是世界上产量最大的水果,巴西是世界上产柑橘最多的国家,其次是中国、美国、墨西哥和西班牙。柑桔黄龙病(Citrus huanglongbing,HLB)是由难人工培养的韧皮杆菌(Cndidatus Liberibacter asiaticus)引起的柑桔毁灭性病害。目前还没有获得该病原菌的纯培养。新近的分子鉴定结果表明黄龙病罹病组织内除存在韧皮杆菌外,还存在其它致病微生物:植原体或伴生微生物,并认为其可能是黄龙病植株症状表现复杂多样的内在原因。本研采用常规分离培养技术与分子鉴定以及基于16S rDNA基因的RFLP(Restriction Fragment Length Polymorphism)法,对柑桔黄龙病植株和健康植株的韧皮部内生菌区系进行比较分析,为研究柑橘植株韧皮部内生细菌与黄龙病菌之间的相互关系奠定基础。
利用分离培养的方法,根据菌落形态以及菌体形态共分离到21株柑桔韧皮部内生细菌,对21株内生细菌进行形态和生理生化测定。经形态学鉴定和序列比对:菌株WJ01,WJ02,WJ03,WJ05,WJ08,WJ12,WJ13,WJ16,WJ17,WJ18,WJ21为芽孢杆菌属(Bacillus sp.),WJ04和WJ14为假单胞菌属(Pseudomonas sp.),WJ06为葡萄球菌属(Staphylococcus sp.),WJ10为考克氏菌(Kocuria sp.)。其中鉴定到种的部分菌株:WJ07:约氏不动杆菌(Acinetobacter johnsonii)、WJ09:粘质沙雷氏菌(Serratia marcescens)、WJ11:乳杆菌属草乳杆菌(Lysinibacillus graminis)、WJ15:微球菌属栖息微球菌(Miccrococcus sedentarius)、WJ19:产左聚糖微杆菌(Microbacterium laevaniformans)、WJ20;极小短小杆菌(Curtobacterium pusillum)的特征。在健康柑橘植株韧皮部中Bacillus sp.、Pseudomonas sp.、Kocuria sp.为优势菌群。在HLB柑橘植株韧皮部中Microbacterium sp.、Bacillus sp.、Pseudomonas sp.和Kocuria sp.为优势菌。且病株与健株相比, WJ19(Microbacterium sp.)的丰富度增加最为明显。
利用16S rDNA-RFLP的方法分析柑桔内生细菌多样性。首先采用的保守性引物分别扩增健康和罹病柑桔韧皮部总内生细菌16S rDNA中的高变区V5、V6、V7、V8及V9,得到的片段长度约为700bp。回收产物进行T-A克隆,健株与病株分别随机挑取了229,228个阳性克隆子,进行PCR检测。提取阳性克隆子的质粒。含有的目的片段的质粒分别用MspⅠ,RsaⅠ,HaeⅢ限制性内切酶进行酶切。鉴别分析各个阳性克隆的酶切图谱类型,对文库中的所有克隆进行分型,不同的类型即是不同的分类操作单位(Operational Taxonomic Unit,OTU)。将不同的OTUs进行测序。测序结果与GenBank中的序列进行同源性比较,结果显示:鉴定出10个属的细菌以及11个不可培养细菌,其中9个OTUs的序列与GenBank中的序列比对相似性≤97%,这些可能是还未经过序列测定过的新菌种。不同属的RFLP OUTs在健株(health plant,HP)与病株(infected plant,IP)的16S rDNA克隆文库中所占的比率不同,分析结果显示:Dyella sp.(HP 27.08%;IP 25%),Enterobacter sp. (HP 3.93%;IP 0%),Serratia marcescens (HP16.16%;IP 18.86%),Enterococcus sanguinicola(HP 3.06%;IP 3.95%),Achromobacter sp.(HP 1.75%;IP 1.32%),Bosea sp. (HP 1.31%;IP 0.44%),Lysinibacillus fusiformis(HP 0%;IP 0.88%),Candidatus Liberibacter asiaticus(HP 0%;IP 3.07%),Bacillus sp.(HP 1.31%;IP 1.31%),Pseudomonas sp(.HP 17.03%;IP 17.54%),Uncultured bacterium clone(HP 28.38%;IP 27.63%)。
常规分离培养与16S rDNA-RFLP的分析方法得到的结果不一致,这说明每种方法都有一定的优势性和局限性;单一使用存在局限性,需要两种方法结合,能够更全面地分析柑桔韧皮部内生细菌的多样性。
常规分离培养与16S rDNA-RFLP的序列分析研究结果表明:健康和罹病柑桔的韧皮部都存在丰富的内生细菌,且这些内生细菌优势菌的种群丰富度与种类存在一定差异。深入研究黄龙病侵染后韧皮部中存在的Microbacterium sp.等不同的细菌菌群与黄龙病菌的相互作用可望发现促生或抑制活性的伴生微生物,为黄龙病病原物的人工培养提供新方法
Citrus is an important economic crop in the world, the major commercial citrus growing areas include southern China, the Mediterranean Basin (including southern Spain), South Africa, Australia, the southernmost United States and parts of South America. In China, Guangxi, Guangdong, Fujian, Sichuan, Hunan, Hubei and Zhejiang provinces are major production areaes, while smaller plantings are present in other provinces. Huanglongbing in China, is probably the one of worst diseases of citrus industry, which caused by a tiny bacterial pathogen. The causative agent is a motile bacterium, Candidatus Liberibacter spp. Field transmission is mainly by Asian citrus psyllids (Sternorrhyncha: Psyllidae, Diaphorina citri).The disease was first described in 1929 and first reported grafting transmission in China in 1956. The African variation was first reported in 1947 in South Africa, where it is still widespread. There is only few reports available about accepted pure culture of the pathogenalthough recently breakthrough in co-culture of HLB causal agents indicated that some company microorganisms are present in theHuanglongbing infected host tissue, and it may causes complex and diverse symptom of Huanglongbing pathogen-Infected citrus. The morphological, physiological and biochemistry characteristics combined with 16S rRNA-RFLP (Restriction Fragment Length Polymorphism) analysis method were applicated for researching the flora of endophytic bacterial populations,especially the microbial diversity in the healthy and Huanglongbing pathogen-infected citrus. All of these aimed to find associated microorganisms of huanglongbing pathogen were investigated.
By the traditional isolation identification and 16S rDNA amplificons methods, 10 genera of bacteria were identified from 21 isolated bacterial populations. The sequences aligned with GenBank database and showed that they were belonged to 10 different genera of bacterium. The morphological and sequence analysis showed that: WJ01, WJ02, WJ03, WJ05, WJ08, WJ12, WJ13, WJ16, WJ17, WJ18, WJ21 belonged to Bacillus sp., WJ04 and WJ14 belonged to Pseudomonas sp., WJ06 belonged to Staphylococcus sp., WJ10 belonged to Kocuria sp. Some strains were also reached the level of species: WJ07, WJ09, WJ11, WJ15, WJ19, WJ20 were respectively consistent with Acinetobacter johnsonii, Serratia marcescens, Lysinibacillus graminis, Miccrococcus sedentarius, Microbacterium laevaniformans, Curtobacterium pusillum. The dominant bacteria in healthy citrus belonged to Bacillus sp., Pseudomonas sp., Kocuria sp.; and in Huanglongbing pathogen-infected citrus were Bacillus sp., Pseudomonas sp., Kocuria halotolerans, Microbacterium sp.
By the PCR-RFLP analysis, two 16S rRNA libraries of Endophytic bacteria were constructed. The total DNA of microorganisms was extracted from the phloem of health and huanglongbing pathogen-infected citrus, and then amplified the 16S rRNA partial sequence: the highly variable region V5, V6, V7, V8 and V9. Primers were designed to generate about a 700 bp of product, corresponding to nucleotides 799–1492 of the 16S rRNA. Purified PCR amplification products were ligated into the pMD19-T cloning vector and transformed into competent Escherichia coli JM109 cells using electric-shock. For T-A cloning, healthy plants and diseased plants randomly picked up 229, 228 positive clones were carried out by PCR. The plasmid, containing the target fragment, were extracted from the positive clonesand used to digested by MspⅠ, RsaⅠ, HaeⅢrestriction enzyme. Those samples with different profiles were considered as different clones and represented a different bacterial strain. A representative from each of the different profile groups observed was sequenced. 16S rRNA sequences were compared with sequence data deposited in GenBank, using the BLAST alignment with sequence data held at GenBank. The results showed that: 29 restriction endonuclease types were detected, 10 genera of bacteria and 8 genera of uncultured bacterium were identified. Different genera of RFLP OUTs in healthy plants (HP) and Infected (IP) plants of the 16S rDNA clone library,the proportion of different analysis results showed that: Dyella sp. (HP 27.08%; IP 25%),Enterobacter sp . (HP 3.93%; IP 0%),Serratia marcescens (HP 16.16%; IP 18.86%),Enterococcus sanguinicola (HP 3.06%; IP 3.95%), Achromobacter sp. (HP 1.75%; IP 1.32%), Bosea sp . (HP 1.31%; IP 0.44%), Lysinibacillus fusiformis (HP 0%; IP 0.88%), Candidatus Liberibacter asiaticus (HP 0%; IP 3.07%), Bacillus sp. (HP 1.31%; IP 1.31%), Pseudomonas sp. (HP 17.03%; IP 17.54%), Uncultured bacterium clone (HP 28.38%; IP 27.63%).
The results showed that: By traditional cultivation, 16S rDNA secquenceing alignment and the PCR-RFLP analysis, there are plenty of microbiota existed in both the healthy and Huanglongbing infected citrus phloem. But the density and species were not exactly same. This suggested that the changes of microbiota flora were maybe assiaciated with the infection of huanglongbing pathogen in citrus phloem. There are a lot of unculturable microorganisms can not be isolated and uncultured andby artificial cultivation method. Therefore more information of microorganism diversity only can be obtained by combining the molecular methods based on the 16S rRNA sequences with traditional culturing methods.
利用分离培养的方法,根据菌落形态以及菌体形态共分离到21株柑桔韧皮部内生细菌,对21株内生细菌进行形态和生理生化测定。经形态学鉴定和序列比对:菌株WJ01,WJ02,WJ03,WJ05,WJ08,WJ12,WJ13,WJ16,WJ17,WJ18,WJ21为芽孢杆菌属(Bacillus sp.),WJ04和WJ14为假单胞菌属(Pseudomonas sp.),WJ06为葡萄球菌属(Staphylococcus sp.),WJ10为考克氏菌(Kocuria sp.)。其中鉴定到种的部分菌株:WJ07:约氏不动杆菌(Acinetobacter johnsonii)、WJ09:粘质沙雷氏菌(Serratia marcescens)、WJ11:乳杆菌属草乳杆菌(Lysinibacillus graminis)、WJ15:微球菌属栖息微球菌(Miccrococcus sedentarius)、WJ19:产左聚糖微杆菌(Microbacterium laevaniformans)、WJ20;极小短小杆菌(Curtobacterium pusillum)的特征。在健康柑橘植株韧皮部中Bacillus sp.、Pseudomonas sp.、Kocuria sp.为优势菌群。在HLB柑橘植株韧皮部中Microbacterium sp.、Bacillus sp.、Pseudomonas sp.和Kocuria sp.为优势菌。且病株与健株相比, WJ19(Microbacterium sp.)的丰富度增加最为明显。
利用16S rDNA-RFLP的方法分析柑桔内生细菌多样性。首先采用的保守性引物分别扩增健康和罹病柑桔韧皮部总内生细菌16S rDNA中的高变区V5、V6、V7、V8及V9,得到的片段长度约为700bp。回收产物进行T-A克隆,健株与病株分别随机挑取了229,228个阳性克隆子,进行PCR检测。提取阳性克隆子的质粒。含有的目的片段的质粒分别用MspⅠ,RsaⅠ,HaeⅢ限制性内切酶进行酶切。鉴别分析各个阳性克隆的酶切图谱类型,对文库中的所有克隆进行分型,不同的类型即是不同的分类操作单位(Operational Taxonomic Unit,OTU)。将不同的OTUs进行测序。测序结果与GenBank中的序列进行同源性比较,结果显示:鉴定出10个属的细菌以及11个不可培养细菌,其中9个OTUs的序列与GenBank中的序列比对相似性≤97%,这些可能是还未经过序列测定过的新菌种。不同属的RFLP OUTs在健株(health plant,HP)与病株(infected plant,IP)的16S rDNA克隆文库中所占的比率不同,分析结果显示:Dyella sp.(HP 27.08%;IP 25%),Enterobacter sp. (HP 3.93%;IP 0%),Serratia marcescens (HP16.16%;IP 18.86%),Enterococcus sanguinicola(HP 3.06%;IP 3.95%),Achromobacter sp.(HP 1.75%;IP 1.32%),Bosea sp. (HP 1.31%;IP 0.44%),Lysinibacillus fusiformis(HP 0%;IP 0.88%),Candidatus Liberibacter asiaticus(HP 0%;IP 3.07%),Bacillus sp.(HP 1.31%;IP 1.31%),Pseudomonas sp(.HP 17.03%;IP 17.54%),Uncultured bacterium clone(HP 28.38%;IP 27.63%)。
常规分离培养与16S rDNA-RFLP的分析方法得到的结果不一致,这说明每种方法都有一定的优势性和局限性;单一使用存在局限性,需要两种方法结合,能够更全面地分析柑桔韧皮部内生细菌的多样性。
常规分离培养与16S rDNA-RFLP的序列分析研究结果表明:健康和罹病柑桔的韧皮部都存在丰富的内生细菌,且这些内生细菌优势菌的种群丰富度与种类存在一定差异。深入研究黄龙病侵染后韧皮部中存在的Microbacterium sp.等不同的细菌菌群与黄龙病菌的相互作用可望发现促生或抑制活性的伴生微生物,为黄龙病病原物的人工培养提供新方法
Citrus is an important economic crop in the world, the major commercial citrus growing areas include southern China, the Mediterranean Basin (including southern Spain), South Africa, Australia, the southernmost United States and parts of South America. In China, Guangxi, Guangdong, Fujian, Sichuan, Hunan, Hubei and Zhejiang provinces are major production areaes, while smaller plantings are present in other provinces. Huanglongbing in China, is probably the one of worst diseases of citrus industry, which caused by a tiny bacterial pathogen. The causative agent is a motile bacterium, Candidatus Liberibacter spp. Field transmission is mainly by Asian citrus psyllids (Sternorrhyncha: Psyllidae, Diaphorina citri).The disease was first described in 1929 and first reported grafting transmission in China in 1956. The African variation was first reported in 1947 in South Africa, where it is still widespread. There is only few reports available about accepted pure culture of the pathogenalthough recently breakthrough in co-culture of HLB causal agents indicated that some company microorganisms are present in theHuanglongbing infected host tissue, and it may causes complex and diverse symptom of Huanglongbing pathogen-Infected citrus. The morphological, physiological and biochemistry characteristics combined with 16S rRNA-RFLP (Restriction Fragment Length Polymorphism) analysis method were applicated for researching the flora of endophytic bacterial populations,especially the microbial diversity in the healthy and Huanglongbing pathogen-infected citrus. All of these aimed to find associated microorganisms of huanglongbing pathogen were investigated.
By the traditional isolation identification and 16S rDNA amplificons methods, 10 genera of bacteria were identified from 21 isolated bacterial populations. The sequences aligned with GenBank database and showed that they were belonged to 10 different genera of bacterium. The morphological and sequence analysis showed that: WJ01, WJ02, WJ03, WJ05, WJ08, WJ12, WJ13, WJ16, WJ17, WJ18, WJ21 belonged to Bacillus sp., WJ04 and WJ14 belonged to Pseudomonas sp., WJ06 belonged to Staphylococcus sp., WJ10 belonged to Kocuria sp. Some strains were also reached the level of species: WJ07, WJ09, WJ11, WJ15, WJ19, WJ20 were respectively consistent with Acinetobacter johnsonii, Serratia marcescens, Lysinibacillus graminis, Miccrococcus sedentarius, Microbacterium laevaniformans, Curtobacterium pusillum. The dominant bacteria in healthy citrus belonged to Bacillus sp., Pseudomonas sp., Kocuria sp.; and in Huanglongbing pathogen-infected citrus were Bacillus sp., Pseudomonas sp., Kocuria halotolerans, Microbacterium sp.
By the PCR-RFLP analysis, two 16S rRNA libraries of Endophytic bacteria were constructed. The total DNA of microorganisms was extracted from the phloem of health and huanglongbing pathogen-infected citrus, and then amplified the 16S rRNA partial sequence: the highly variable region V5, V6, V7, V8 and V9. Primers were designed to generate about a 700 bp of product, corresponding to nucleotides 799–1492 of the 16S rRNA. Purified PCR amplification products were ligated into the pMD19-T cloning vector and transformed into competent Escherichia coli JM109 cells using electric-shock. For T-A cloning, healthy plants and diseased plants randomly picked up 229, 228 positive clones were carried out by PCR. The plasmid, containing the target fragment, were extracted from the positive clonesand used to digested by MspⅠ, RsaⅠ, HaeⅢrestriction enzyme. Those samples with different profiles were considered as different clones and represented a different bacterial strain. A representative from each of the different profile groups observed was sequenced. 16S rRNA sequences were compared with sequence data deposited in GenBank, using the BLAST alignment with sequence data held at GenBank. The results showed that: 29 restriction endonuclease types were detected, 10 genera of bacteria and 8 genera of uncultured bacterium were identified. Different genera of RFLP OUTs in healthy plants (HP) and Infected (IP) plants of the 16S rDNA clone library,the proportion of different analysis results showed that: Dyella sp. (HP 27.08%; IP 25%),Enterobacter sp . (HP 3.93%; IP 0%),Serratia marcescens (HP 16.16%; IP 18.86%),Enterococcus sanguinicola (HP 3.06%; IP 3.95%), Achromobacter sp. (HP 1.75%; IP 1.32%), Bosea sp . (HP 1.31%; IP 0.44%), Lysinibacillus fusiformis (HP 0%; IP 0.88%), Candidatus Liberibacter asiaticus (HP 0%; IP 3.07%), Bacillus sp. (HP 1.31%; IP 1.31%), Pseudomonas sp. (HP 17.03%; IP 17.54%), Uncultured bacterium clone (HP 28.38%; IP 27.63%).
The results showed that: By traditional cultivation, 16S rDNA secquenceing alignment and the PCR-RFLP analysis, there are plenty of microbiota existed in both the healthy and Huanglongbing infected citrus phloem. But the density and species were not exactly same. This suggested that the changes of microbiota flora were maybe assiaciated with the infection of huanglongbing pathogen in citrus phloem. There are a lot of unculturable microorganisms can not be isolated and uncultured andby artificial cultivation method. Therefore more information of microorganism diversity only can be obtained by combining the molecular methods based on the 16S rRNA sequences with traditional culturing methods.
引文
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[48] Ordentlich A, Elad Y, Chet I. The role of chitin ase of Serratia marcescens in biocontrol of Sclerotium Rolfsii[J]. Phytopathology, 1988, 78: 84-88.
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[51] Schneider H. Anatomy of greening diseased sweet orange shoots [J]. Phytopathology, 1968, 58: 1155–1160.
[52] Sechler A, Schuenzel E L, Cooke P, Donnua S, Thaveechai N, Postnikova E, Stone A L, Schneider W L, Damsteegt V D, Schaad N W. Cultivation of‘Candidatus Liberibacter asiaticus’,‘Ca. L. africanus’, and‘Ca. L. americanus’Associated with Huanglongbing[J]. Phytopathology. 2009, 99(5): 480-486.
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[61] Tian Y K,Wang C H,Zhang J P,Dai H Y,Gu Y,Duan F M. Optimization of factors affecting RAPD amp lification in app le genome[J]. J Laiyang Agri Coll,2003,20 (3) : 157-161.
[62] Torsvik V,Ovreas L. Microbial diversity and function in soil: from genes to ecosystems. Curr. Opin. Microbiol, 2002, 5: 240-245.
[63] Vergin K L, Rappe′M S, Giovannoni S J. Streamlined method to analyze 16S rRNA gene clone libraries [J]. BioTechniques, 2001, 30: 938-944.
[64] Wang Z K, Yin Y P, Yuan Q, Peng G X, Xia Y X. Development and application of molecular-based diagnosis for‘Candidatus Liberibacter asiaticus’, the causal pathogen of citrus huanglongbing[J]. Plant Pathology, 2006, 55: 630-638.