两株棉花立枯病拮抗菌MH1和MH25的筛选与鉴定

从棉花根际分离了1277个细菌分离物,以棉花立枯病病原真菌立枯丝核菌(Rhizoctonia solani Kuhn)为靶标菌,通过平板对峙法获得25个具有拮抗性能的分离物,其中MH1和MH25具有较强的拮抗性能,且拮抗性能稳定,具有较好的生防潜力.经过形态观察、生理生化特征分析及16S rDNA序列分析,MH1为短芽孢杆菌(Brevibacillus brevis),MH25为枯草芽孢杆菌(Bacillus subtilis).MH1和MH25的16S rDNA序列在GenBank中注册号分别为:EF488102,EF488103.     

枯草芽孢杆菌MH25 Iturin A操纵子的克隆与分析

根据已知非核糖体肽合成抗生素操纵子的保守序列设计引物,从对棉花立枯病有很好拮抗作用的枯草芽孢杆菌(Bacillus subtilis)MH25菌株中克隆相关操纵子.获得了枯草芽孢杆菌MH25的一个非核糖体肽合成抗生素操纵子序列,其包括4个ORF(ORF1,ORF2,OKF3,ORF4),与枯草芽孢杆菌RB14的ituD,ituA,tiuB和ituC的同源性分别为99%,98.70%,98.99%和99.48%,4个ORF编码的氨基酸序列与ItuD,ItuA,ItuB,ItuC的相似性分别为98%,98.54%,98.69%和98%.然后将4个ORF分别进行结构域分析,ORF3的14 779～14 963序列与ituB相对应区域的相似性为86.24%.该操纵子的启动子区为TATACACA-16bp-TAGGAT,与σA-10和-35(TTGACA-17bp-TATAAAT)不同.枯草芽孢杆菌MH25的Iturin A操纵子序列已在GenBank中注册,登陆号为EU263005.     

药用植物内生芽孢杆菌的多样性和系统发育研究

[目的]了解药用植物内生芽孢杆菌的生物多样性.[方法]采用数值分类、16S rDNA PCR RFLP、BOX-PCR指纹图谱和16S rDNA序列分析技术对分离于几种药用植物的内生芽孢杆菌和已知参比菌株进行表型、遗传多样性及系统发育研究.[结果]供试菌株在数值分类聚类分析中在84%的相似水平上产生13个表观群.16S rDNAPCR-RFLP分析表明供试菌株表现出丰富的遗传多样性.BOX-PCR指纹图谱分析进一步证明药用植物的内生芽孢杆菌的基因组也具有多样性,聚群的结果与数值分类有较好一致性.用软件在Genbank中进行所得序列的同源性检索,并构建系统发育树.由16S rDNA序列分析可知,供试的代表菌株SCAU11与球形芽孢杆菌(Bacillus sphaericus)亲缘关系最近,SCAU78和SCAU25为枯草芽孢杆菌(Bacillus subtilis)的两个亚种,代表菌株SCAU39与巨大芽孢杆菌(Bacillus megaterium)的亲缘关系最近.[结论]研究结果表明药用植物内生芽孢杆菌具有明显的表型和遗传多样性.     

三株芸豆叶际拮抗细菌的筛选与鉴定

以芸豆锈病病原菌[Uromyces appendiculatus(Pers.)Ung.]为指示菌,从芸豆叶际中筛选到3株具有明显拮抗效果的细菌,编号为SS2,L14b,NEW2。经过形态学观察,生理生化测定,16S rDNA序列及系统发育分析,初步鉴定SS2为短短芽孢杆菌(Brevibacillus brevis),L14b与NEW2为枯草芽孢杆菌(Bacillus subtilis)。SS2,L14b,NEW2的16S rDNA序列的Gen-Bank登录号分别为EU771078、EU771076和EU771079。     

水浮莲中一株西瓜枯萎病拮抗菌的分离、筛选及鉴定

从水浮莲(Pistia stratiotes L.)的叶中分离出1株对西瓜枯萎病有明显拮抗作用的内生细菌XJPL-YB-26, 其发酵液上清在280 nm处有最大紫外吸收峰。利用软件Primer 6.0 设计16S rDNA引物并对其基因组DNA进行扩增并测序得到XJPL-YB-26的部分16S rDNA序列, GenBank接收号为EU251191。经Blastn调出与菌株16S rDNA同源的序列, 并用软件MEGA 3.1按Neighbor- Joining方法构建16S rDNA系统发育树。菌株XJPL-YB-26与AB271744处于同一分支, 相似性为99%, 最终鉴定为枯草芽孢杆菌Bacillus subtilis。     

水浮莲中-株西瓜枯萎病拮抗菌的分离、筛选及鉴定

从水浮莲(Pistia stratiotes L.)的叶中分离出1株对西瓜枯萎病有明显拮抗作用的内生细菌XJPL-YB-26,其发酵液上清在280 nm处有最大紫外吸收峰.利用软件Primer 6.0设计16S rDNA引物并对其基因组DNA进行扩增并测序得到XJPL-YB-26的部分16S rDNA序列.GenBank接收号为EU251191.经 Blastn调出与菌株16S rDNA同源的序列,并用软件MEGA 3.1按Neighbor-Joining方法构建16S rDNA系统发育树.菌株XJPL-YB-26与AB271744处于同一分支,相似性为99%,最终鉴定为枯草芽孢杆菌Bacillus subtilis.     

中药中耐辐射微生物的分离与鉴定

从中成药(金锁丸)中分离获得一种耐辐射的细菌。经过形态观察、生理生化特征分析及16 S rDNA碱基序列测定和同源性分析,鉴定该菌株为弯曲芽孢杆菌(Bacillus flexus),该菌株的16 S rDNA序列已在Gen-Bank中注册,登录号为AB021185.1。     

拮抗菌株Kc-t99的鉴定及其抑菌活性研究

通过传统分类与分子生物学技术相结合的方法对从京郊菜园土壤中分离筛选的拮抗菌株Kc-t99进行鉴定,并采用生物测定的方法评价其抑菌活性.结果表明,菌株Kc-t99的形态学特征和生理生化特性与枯草芽孢杆菌基本一致,其16S rDNA序列与GenBank中已鉴定的枯草芽孢杆菌的16S rDNA序列同源性高达98.06%,据此初步确定其为枯草芽孢杆菌.抑菌试验表明该菌株对供试的5种病原真菌和4种细菌均具抑菌活性,其中对甘蓝枯萎病菌、黄瓜角斑病菌和桃褐腐病菌抑制作用显著.     

变质豆浆中腐败微生物的分离与初步鉴定

从5种变质豆浆中分离得到3株腐败菌株S1、S2和S3。3株菌均能在1×105Pa、30min杀菌条件下和添加300mg/kgNisin杀菌条件下存活。对分离菌株进行了菌落形态、生理生化特征和16SrDNA序列分析,鉴定结果表明3株菌均为革兰氏阴性细菌,分别为地衣芽孢杆菌(Bacillus licheniformis)、短小芽孢杆菌(Bacillus pumilus)和短芽孢杆菌(Brevibacillus borstelensis),GenBank登录号为EF439666~EF439668。     

具细菌群体感应抑制活性海洋细菌的筛选鉴定

目的:从海洋环境中筛选对细菌群体感应有抑制作用的活性菌株,为以致病菌群体感应系统为靶点的新型疗法提供新的药用资源。方法:从海水中分离纯化细菌菌株,采用根癌农杆菌平板筛选模型筛选细菌群体感应抑制活性细菌,对筛选出的海洋细菌进行生理生化和16S rDNA序列测定,根据《伯杰氏手册》进行菌种分类鉴定。结果:从217株海洋细菌中筛选出1株能显著抑制细菌群体感应效应的海洋细菌Y2,该海洋细菌具有蜡样芽孢杆菌(Bacillus cereus)的典型特征,其16S rDNA序列与GenBank中蜡样芽孢杆菌16S rDNA的部分序列有100%的同源性。结论:海洋环境中也存在具有抑制细菌群体感应活性的微生物。     

Influence of the Presence of B Chromosomes on DNA Damage in Crepis capillaris

The sensitivity of different plant species to mutagenic agents is related to the DNA content and organization of the chromatin, which have been described in ABCW and bodyguard hypotheses, respectively. Plant species that have B chromosomes are good models for the study of these hypotheses. This study presents an analysis of the correlation between the occurrence of B chromosomes and the DNA damage that is induced by the chemical mutagen, maleic hydrazide (MH), in Crepis capillaris plants using comet assay. The presence of B chromosomes has a detectable impact on the level of DNA damage. The level of DNA damage after MH treatment was correlated with the number of B chromosomes and it was observed that it increased significantly in plants with 3B chromosomes. We did not find evidence of the protective role from chemical mutagens of the constitutive heterochromatin for euchromatin in relation to DNA damage. The DNA damage involving the 25S rDNA sequences was analyzed using the comet-FISH technique. Fragmentation within or near the 25S rDNA involved the loci on the A and B chromosomes. The presence of B chromosomes in C. capillaris cells had an influence on the level of DNA damage that involves the 25S rDNA region.     

Elongation factor 1-alpha sequences do not support an early divergence of the Acoela

The phylogenetic position of the Acoela is a key problem in the understanding of metazoan evolution. Recent studies based on 18S ribosomal DNA (rDNA) sequences have placed the Acoela in an extremely basal position as the sister group to all other extant triploblastic animals, suggesting that the phylum Platyhelminthes is polyphyletic. In order to test the results obtained with 18S rDNA, we sequenced elongation factor 1-alpha (EF1a) for the acoel Convoluta roscoffensis and five species of Turbellaria (two polyclads, Leptoplana tremellaris, and Prostheceraeus vittatus, and three triclads, Crenobia alpina, Schmidtea polychroa, and Girardia tigrina). Phylogenetic analyses of EF1a sequences show that the acoel sequences branch within the Platyhelminthes, in opposition to the 18S rDNA data. Moreover, comparison of the central variable region of EF1a shows similar sequence signatures between C. roscoffensis and the three triclad species. Although EF1a sequences fail to prove the monophyly of the phylum Platyhelminthes, they do not confirm the early divergence of the Acoela.     

Diversity and geographical distribution of rhizobia associated with <Emphasis Type="Italic">Lespedeza</Emphasis> spp. in temperate and subtropical regions of China

Eighty-eight root-nodule isolates from Lespedeza spp. grown in temperate and subtropical regions of China were characterized by a polyphasic approach. Nine clusters were defined in numerical taxonomy and SDS-PAGE analysis of whole cell proteins. Based upon further characterizations of amplified 16S rDNA restriction analysis (ARDRA), PCR-based restriction fragment length polymorphism of ribosomal IGS, 16S rDNA sequence analysis and DNA-DNA hybridization, these isolates were identified as Bradyrhizobium japonicum, B. elkanii, B. yuanmingense, Mesorhizobium amorphae, M. huakuii, Sinorhizobium meliloti and three genomic species related to B. yuanmingense, Rhizobium gallicum and R. tropici. The Bradyrhizobium species and R. tropici-related rhizobia were mainly isolated from the subtropical region and the species of Mesorhizobium, S. meliloti and R. gallicum-related species were all isolated from the temperate region. Phylogenetic analyses of nifH and nodC indicated that the symbiotic genes of distinct rhizobial species associated with Lespedeza spp. might have different origins and there was no evidence for lateral gene transfer of symbiotic genes. The results obtained in the present study and in a previous report demonstrated that Lespedeza spp. are nodulated by rhizobia with diverse genomic backgrounds and these Lespedeza-nodulating rhizobia were not specific to the host species, but specific to their geographic origins. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users. GenBank sequence accession numbers: The GenBank accession numbers were EF61095 through EF061114 and EF051240 for acquired 16S rDNA sequences; EF153395 through EF153402 for nifH sequences; and EF153403 through EF153410 for nodC sequences.     

Theoretical and Practical Approaches to Evaluate Suitable Primer Sets for the Analysis of Soil Fungal Communities

Six published fungal specific primer sets (NS1/NS2, SSU‐0817/SSU11‐96, SSU‐0817/SSU‐1536, EF4/EF3, EF4/fung5 and FR1/FF390) were examined for their applicability to the analysis of soil fungal communities using bioinformatic tools as well as real PCR systems. Virtual primer matching for EF4/EF3 and EF4/fung5 revealed good matching with zygomycetous, ascomycetous and basidiomycetous 18S rDNA database entries. Whereas primer EF4/EF3 had no cross matches in the rDNA databases for plant and invertebrate, primer EF4/fung5 gave one signal with the corresponding database. Similar results were obtained for the primer set SSU‐0817/SSU‐1536. Two matches with plant rDNAs and 22 or 12 matches with the invertebrate database could be identified for the primer sets SSU‐0817/SSU‐1196 and FR1/FF390, respectively. Primer pair NS1/NS2 showed only a 70% match with fungal 18S rDNA sequences, but a 75% to 90% match with non‐fungal sequences. Alignments of 2000 eukaryotic sequences using “ARB” confirmed that PCR fragments obtained by the primer sets EF4/EF3, EF4/fung5, SSU‐0817/SSU‐1536 and FR1/FF390 were supposed to include hypervariable regions (V4, V7, V9), whereas the others included regions which were more phylogenetically conserved. Practical PCR approaches affirmed fungal specificity as predicted by virtual primer matching for EF4/EF3, EF4/fung5 and FR1/FF390. However FR1/FF390 amplified only 60% of the fungal samples under investigation. All other primer sets amplified fungal as well as non‐fungal samples.     

高产β-葡萄糖苷酶N1菌株的分类鉴定

从纳豆中分离纯化得到1株β-葡萄糖苷酶高产菌株,命名为N1菌,利用16S rDNA分析对该菌作了系统进化鉴定.首先提取N1菌基因组DNA,根据不同种属细菌的16S rDNA序列两端的保守性设计通用引物,对N1菌16S rDNA片断进行PCR扩增,对扩增得到的目标片段测序,利用BLAST对测序结果进行比对,构建系统进化树进行分析,初步确定该菌属于枯草芽孢杆菌.研究表明以16S rDNA分析对该菌鉴定完全可行.     

Physical mapping of rDNA genes establishes the karyotype of almond

The localisation of ribosomal RNA genes on chromosomes of almond (Prunus amygdalus, 2n = 16) was studied by fluorescence in situ hybridisation. Simultaneous double-colour hybridisation with both 18S–5.8S–25S and 5S rDNA probes demonstrated that all chromosomes can be identified. In spite of the small size, differences in length between chromosomes that hybridised with the same rDNA probe as well as between chromosomes without hybridisation signal are apparent. Chromosomes were ordered in the karyotype according to their length. The 18S-5.8S-25S rDNA genes were detected in subdistal positions of chromosomes 2, 3, and 8. Sites located on chromosomes 2 and 3 carry a higher number of repeats than the site of chromosome 8. The 5S rDNA genes were found proximally located on chromosomes 5 and 7, the signal on chromosome 5 showing higher intensity than the signal on chromosome 7. Chromosomes 1, 4, and 6 show no hybridisation signal.     

脱氮除硫菌株的分离鉴定和功能确认

从长期稳定运行的脱氮除硫反应器污泥中,分离获得两株具有脱氮除硫功能的芽孢杆菌。经形态观察、生理试验和16SrDNA序列比对,将两菌株归入芽孢杆菌属,菌株CB归类于Bacillus pseudofirmus,菌株CS则与Bacillus hemicellulosilytus和Bacillus halodurans最为接近。以Biolog板检测,菌株CB的基质多样性不明显,菌株CS则可利用Biolog板中多种碳源。菌株CB和菌株CS都能以硝酸盐氧化硫化物,其中菌株CB对硝酸盐、硫化物的转化能力大于CS,菌株CB对硝酸盐的亲和力也大于菌株CS。     

Metschnikowia bicuspidata and Enterococcus faecium co-infection in the giant freshwater prawn Macrobrachium rosenbergii

In May 2001, an epizootic yeast and bacterial co-infection in the giant freshwater prawn Macrobrachium rosenbergii occurred in Taiwan causing a cumulative mortality of 25%. The diseased prawns had a yellowish-brown body color, milky hemolymph, opaque, whitish muscles, and were approximately 7 mo old with total lengths ranging from 8 to 10 cm. Histopathological examination showed marked edema, yeast infiltration, and necrotic lesions with inflammation in the muscles, hepatopancreas and other internal organs. We isolated 2 pathogens from the diseased prawns, one was a yeast (AOD081MB) and the other a gram-positive coccus (AOD081EF). The gram-positive coccus was identified as Enterococcus faecium by the API 20 Strepsystem, conventional biochemical tests, and it had 99% 16S rDNA sequence identity (GenBank Accession Number AJ276355) to E. faecium (GenBank Accession Number AF529204). The sequence of a PCR product from the D1/D2 domain of 26S rDNA (GenBank Accession Number AF529297) from the yeast gave 99% sequence identity to Metschnikowia bicuspidata (GenBank Accession Number U44822). Experimental infections with these isolates produced gross signs and histopathological changes similar to those observed in the naturally infected prawns. The lethal doses (LD50) for isolate E. faecium AOD081EF, M. bicuspidata AOD081MB and the co-infection were 4.7 x 10(4), 2.6 x 10(2), and 2.4 x 10(2) colony-forming units prawn(-1), respectively. This is the first report of a confirmed co-infection of M. bicuspidata and E. faecium in prawn aquaculture.     

Endophytic bacterial flora in root and stem tissues of black pepper (Piper nigrum L.) genotype: isolation, identification and evaluation against Phytophthora capsici

Aim:  To isolate and identify black pepper ( Piper nigrum L) associated endophytic bacteria antagonistic to Phytophthora capsici causing foot rot disease.
Methods and Results:  Endophytic bacteria (74) were isolated, characterized and evaluated against P. capsici . Six genera belong to Pseudomonas spp (20 strains), Serratia (1 strain), Bacillus spp. (22 strains), Arthrobacter spp. (15 strains), Micrococcus spp. (7 strains), Curtobacterium sp. (1 strain) and eight unidentified strains were isolated from internal tissues of root and stem. Three isolates, IISRBP 35, IISRBP 25 and IISRBP 17 were found effective for Phytophthora suppression in multilevel screening assays which recorded over 70% disease suppression in green house trials. A species closest match (99% similarity) of IISRBP 35 was established as Pseudomonas aeruginosa ( Pseudomonas EF568931), IISRBP 25 as P. putida ( Pseudomonas EF568932), and IISRBP 17 as Bacillus megaterium ( B. megaterium EU071712) based on 16S rDNA sequencing.
Conclusion:  Black pepper associated P. aeruginosa , P. putida and B. megaterium were identified as effective antagonistic endophytes for biological control of Phytophthora foot rot in black pepper.
Significance and Impact of the Study:  This work provides the first evidence for endophytic bacterial diversity in black pepper stem and roots, with biocontrol potential against P. capsici infection.