新疆野生胀果甘草内生细菌多样性的非培养初步分析

摘要：【目的】了解新疆野生甘草内生细菌多样性,为开发新的微生物资源奠定基础。【方法】采用改进的CTAB (十六烷基三甲基溴化铵)法提取新疆野生胀果甘草根部总DNA,利用细菌16S rDNA 基因通用引物对甘草总DNA 进行16S rDNA 基因扩增,构建甘草内生细菌16S rDNA基因文库;挑选具有不同酶切图谱的克隆进行测序、比对并构建16S rDNA 基因系统发育树。【结果】构建的甘草内生细菌16S rDNA基因文库中, 150个克隆分属于32个不同的分类单元,Blast结果表明大部分克隆与已知细菌的16S rDNA基因序列相似性较高,分别归属于变形杆菌门(Proteobacteria)的alpha、gamma亚群,厚壁菌门(Firmicutes),放线菌门(Actinobacteria),拟杆菌门(Bacteroidetes)中的鞘脂菌属(Sphingobium),叶杆菌属(Phyllobacterium),生丝单胞菌属(Hyphomonas),土壤杆菌属(Agrobacterium)等14个属, 其中26%的克隆与已知细菌16S rDNA 基因相似性小于96%,可能代表新的分类单元.【结论】甘草内生细菌多样性丰富且存在尚未被认识的新物种。     

黑龙江大豆胞囊线虫胞囊可培养细菌多样性

【目的】了解黑龙江省大豆田大豆胞囊线虫胞囊可培养细菌的多样性。【方法】运用稀释平板法和16SrDNA基因序列的系统发育分析对胞囊可培养细菌多样性进行研究。【结果】用NA培养基从胞囊上分离90株具有不同菌落形态的细菌。16S rDNA序列分析结果表明:90株菌株分属于7个属22个种。46株属于变形菌门γ亚群(Gammaproteobacteria),32株属于厚壁菌门(Firmicutes),10株属于变形菌门β亚群(Betaproteobacteria),2株属于变形菌门ɑ亚群(Alphaproteobacteria)。假单胞菌属(Pseudomonas)和芽孢杆菌属(Bacillus)为优势菌属。【结果】黑龙江省大豆胞囊线虫胞囊中存在丰富的细菌物种多样性,这些细菌对大豆胞囊线虫可能具有一定的生理生态作用。     

红豆杉免培养内生细菌多样性的初步研究

【目的】了解红豆杉(Taxus chinensis)内生细菌的组成及多样性。【方法】提取红豆杉组织总DNA,选用细菌通用引物799F和1492R对总DNA进行16S rDNA特异性扩增,构建红豆杉内生细菌16S rDNA克隆文库,对阳性克隆进行PCR-RFLP(限制性内切酶片段长度多态性)分析,并对酶切带谱不同的菌液进行测序,构建系统发育树。【结果】根据酶切带谱分析和测序结果的不同,将随机挑取的158个阳性克隆归为26个不同的可操作分类单元(OUTs),系统发育分析表明这些克隆序列分别属于变形菌门(Proteobacteria,包含Alpha、Beta、Gamma、Delta亚群)、厚壁菌门(Firmicutes)、放线菌门(Actinobacteria)和拟杆菌门(Bacteroidetes)4个门。其中,变形菌门(Proteobacteria,占克隆总数的58.86%)为最优势类群。序列比对结果表明这些克隆序列分别与已报道的20个属具有较高的相似性。此外,还有一个OUTs在系统发育树上形成独立分支且未能确定其分类。【结论】红豆杉内生细菌多样性丰富,并且可能存在新的分类单元。     

棉花内生细菌数量动态及其对棉花黄、枯萎病菌的拮抗作用

摘要：【目的】了解棉花内生细菌数量动态,从棉花中获得拮抗黄、枯萎病菌的内生细菌资源【方法】对棉花根、茎、叶表面灭菌,采用稀释平板法分离棉花内生细菌;通过对峙培养法体外鉴定分离的棉花内生细菌对棉花黄、枯萎病菌的拮抗作用,并对拮抗棉花黄、枯萎病菌的内生细菌16S rDNA序列进行了分析【结果】棉花根中内生细菌的数量显著高于茎、叶;根的苗期总体内生细菌数量低于开花期、吐絮期,茎、叶中的内生细菌数量在不同生育期呈现一定的波动性,但趋势性不明显;6个棉花品种根中内生细菌平均数量差异并不显著,但茎、叶中内生细菌数量不同品种间呈现一定程度差异。平板对峙鉴定显示:棉花根中具有较高比例的拮抗棉花黄、枯萎病菌的内生细菌,拮抗强致病落叶型黄萎病菌（V107）的内生细菌比例不仅低于枯萎病菌（F108）,而且低于非落叶型黄萎病菌（V396）。同时拮抗棉花黄、枯萎病菌的内生细菌有44株,16S rDNA分子序列分析表明：这些拮抗内生细菌的类群包括了两个门(变形杆菌门、拟杆菌门)8个属,其中10个菌株与已报道菌株相似性<97%,可能是新的种（属）,优势种群为肠杆菌属（18株）、泛菌属（15株）。【结论】棉花的品种、生育期与器官影响棉花内生细菌数量;棉花拮抗黄、枯萎病菌的内生细菌具有优势种群,且具多样性。     

16S rDNA克隆文库法探索转基因香石竹对土壤细菌群落的影响

【目的】通过研究转基因香石竹对土壤细菌群落的影响,为转基因香石竹的环境安全性评价提供依据。【方法】通过构建16S rDNA克隆文库,分析种植转基因和非转基因香石竹的土壤中细菌的群落结构组成。【结果】转基因和非转基因香石竹土壤中,共有的菌群有变形菌门(Proteobacteria)、浮霉菌门(Planctomycetes)、酸杆菌门(Acidobacteria),其中α-变形菌门、β-变形菌门、浮霉菌门为优势菌群;而在放线菌门(Actinobacteria)、疣微菌门(Verrucomicrobia)及未培养菌(Uncultured bacterium clone)等菌群存在部分差异。【结论】通过16S rDNA克隆文库方法揭示了转基因香石竹的土壤中细菌多样性十分丰富,其栽培对土壤细菌群落结构影响有限。     

普洱地区茶叶内生细菌与根际土壤细菌群落结构分析

【目的】茶叶内生细菌、根际土壤细菌在普洱茶的发酵中起着重要的作用,还可以促进茶树生长,诱导茶树抗病性。研究其群落结构组成及相互关系可为微生物资源开发利用提供理论依据。【方法】本研究以普洱地区茶树叶片和根际土壤为材料,采用高通量测序技术,对茶叶及根际土壤细菌的16S核糖体RNA基因(16S rRNA)进行测序,比较分析茶叶与根际土壤细菌的群落结构组成。【结果】结果表明,茶叶内生细菌主要由76个属组成,归属于9个门。相对丰度较高的门是厚壁菌门(Firmicutes)、变形菌门(Proteobacteria)、拟杆菌门(Bacteroidetes)、放线菌门(Actinobacteria)。相对丰度较高的属为乳杆菌属(Lactobacillus)、布劳特氏菌属(Blautia)、粪杆菌属(Faecalibacterium)、假单胞菌属(Pseudomonas)、红球菌属(Rhodococcus)、拟杆菌属(Bacteroide)和普雷沃氏菌属(Prevotella)。根际土壤细菌主要由198个属组成,归属于10个门,相对丰度较高的门是变形菌门(Proteobacteria)、酸杆菌门(Aci...     

对枯萎病不同抗性的香蕉品种的内生细菌多样性及群落结构

【目的】了解抗感品种香蕉植株中内生细菌与香蕉枯萎病间的关系,从而为香蕉枯萎病的发生与防控提供一定的理论基础。【方法】通过基于16S rRNA的末端标记限制性片段长度多态性技术(Terminal restriction fragment length polymorphism,T-RFLP)分析健康和感枯萎病香蕉植株不同品种各组织,以及香蕉植株不同发病时间根组织中内生细菌的多样性和群落结构。【结果】抗病品种"农科一号"植株根、假茎、叶片各组织中内生细菌的种类基本都比感病品种"巴西蕉"的相应组织中的要丰富。感枯萎病香蕉植株根、假茎、叶片各组织中内生细菌种类基本都比健康植株各组织的丰富。在植株发病前、发病初期、再到发病后一个月的不同时期,对于抗病品种而言,其内生细菌的多样性都基本保持稳定,而感病品种则变化幅度较大。同时发现不同品种不同组织的内生细菌的优势种群有所不同,且不同品种健康和发病植株都存在一些特有优势种群。【结论】香蕉抗病品种比感病品种植株中内生细菌的多样性更丰富且更稳定;感枯萎病植株中的内生细菌种类比健康的丰富;而且不同抗性品种中健康和感病植株内的优势种群存在明显差异。     

南京小龙山钾矿区植物根际可培养细菌的遗传多样性分析

摘要:【目的】矿区优势植物可培养细菌生物多样性研究将有助于了解植物根际细菌与矿物,植物根系相互作用及对矿物风化和土壤形成的重要影响。【方法】采用纯培养法分离南京小龙山废钾矿区野生植物野塘蒿,千金子和栽培植物甘薯根内与根周围土壤的可培养细菌, 通过16S rDNA限制性酶切多态性分析(amplified rDNA restriction analysis, ARDRA)和16S rDNA序列分析研究了可培养细菌的多样性。【结果】分离纯化到60株具不同菌落形态的可培养细菌, 在60%相似水平上可分为18个OTU. 19株代表菌株分别属于3个门, 10个科, 11个属。多数菌株属于变形菌门(α-proteobacteria, 4株, 21.1%; β-proteobacteria, 2株, 10.5%; γ-proteobacteria, 6株, 31.6%)。假单胞菌属(Pseudomonas), 泛菌属(Pantoea)和根瘤菌属(Rhizobium)为优势种群。【结论】小龙山废矿区优势植物根围具有丰富的微生物种群多样性。     

携带松材线虫对松墨天牛成虫肠道和气管细菌的影响

【目的】探究被松墨天牛Monochamus alternatus携带的松材线虫Bursaphelenchus xylophilus对松墨天牛肠道和气管细菌的影响。【方法】野外采集携带松材线虫的松墨天牛成虫后,取出完整肠道和气管进行细菌总DNA抽提后进行16S rDNA基因测序并拼接,并利用生物信息学方法分析松墨天牛成虫肠道和气管细菌组成、结构、丰度和多样性。【结果】携带松材线虫的松墨天牛成虫肠道和气管细菌菌群共检测到15门26纲66目110科201属296种,可操作分类单元(operational taxonomic unit, OTU)数目为444。携带松材线虫的松墨天牛成虫比未携带松材线虫的松墨天牛成虫肠道优势细菌菌群变化不显著,均为变形菌门(Proteobacteria)肠杆菌目(Enterobacterales);携带松材线虫的松墨天牛成虫气管优势细菌菌群为变形菌门肠杆菌目,未携带松材线虫的松墨天牛成虫气管优势细菌菌群为厚壁菌门(Firmicutes)乳杆菌目(Lactobacillales)。携带松材线虫的松墨天牛成虫较未携带松材线虫的松墨天牛成虫气管细菌多样性和丰度升高,细...     

新疆玛纳斯热气泉免培养土壤细菌多样性分析

【目的】了解新疆玛纳斯热气泉土壤免培养细菌群落组成及多样性。【方法】采用免培养法直接从土壤样品中提取总DNA,利用细菌通用引物对土壤总DNA进行16S rDNA扩增,构建细菌16SrDNA文库。使用HaeⅢ限制性内切酶对阳性克隆进行限制性片段长度多态性分析(restriction fragment length polymorphism,RFLP),挑选具有不同酶切图谱的克隆进行测序、比对并构建16SrDNA系统发育树。【结果】从土壤细菌16S rDNA文库中随机挑选了170个阳性克隆,共得到29个不同的分类单元(operational taxonomic unit,OTU)。系统发育分析归为6个门:酸杆菌门(Acidobacteria)、放线菌门(Actinobacteria)、拟杆菌门(Bacteroidetes)、厚壁菌门(Firmicutes)、变形菌门(Proteobacteria)和浮霉菌门(Planctomycetes)。其中厚壁菌门(Firmicutes)为绝对优势类群,占整个细菌文库的71%。29个OTUs中有14条序列与GenBank中相关序列的相似性低于97%(序列长度约1.5kb),占序列总数的48%。【结论】热气泉土壤细菌种群多样性较低,但存在大量潜在细菌新种。     

Photosynthetic responses of soybean to soybean aphid (Homoptera: Aphididae) injury

The soybean aphid, Aphis glycines Matsumara, was discovered in the United States in the summer of 2000. Since that initial discovery, the aphid has spread across northern soybean production regions. In 2001, we examined the physiological responses of soybeans to low aphids densities (fewer than 50 aphids/leaf). In this study, we determined photosynthetic rates, leaf fluorescence responses, and photosynthetic responses to variable carbon dioxide and light levels. In addition, analyses for chlorophyll content and stable carbon isotope ratios were used to differentiate potential differences in stomatal versus mesophyll limitations to photosynthesis. We observed rate reductions of up to 50% on infested leaflets, including lealets with no apparent symptoms of aphid injury (such as chlorosis). Differences in fluorescence data indicated that photoelectron transport was not impaired. These results indicate that substantial physiological impact on soybean is possible even at low aphid densities. Also, the conventional view of aphid injury acting through reductions in chlorophyll content and light-harvesting reactions of photosynthesis is not supported by our findings in this system.     

Temperature influences the expression of resistance of soybean (Glycine max) to the soybean aphid (Aphis glycines)

Resistance of plants to arthropods may be lost at low or high temperatures. I tested whether the relative resistance of five genotypes of soybean, Glycine max (L.) Merr., to three isolates of the soybean aphid, Aphis glycines Matsumura, was influenced by three temperatures, 14, 21 and 28°C, in no‐choice tests in the laboratory. The interaction between temperature and the genotype of soybean influenced the population sizes of two isolates of A. glycines. Two genotypes of soybean, LD05‐16611 and PI 567597C, which usually are resistant to isolate 1 and 3, became susceptible: LD05‐16611 at the low temperature and PI 567597C at the high temperature. The genotypes PI 200538 and PI 567541B usually are susceptible to isolate 3 but were resistant at 21 and 28°C. I can only speculate as to the reason why temperature influences resistance of some genotypes of soybean to A. glycines: A. glycines may be directly influenced by temperature or indirectly influenced by changes in the host plant. Nevertheless, my results suggest that temperature may be one factor that influences the expression of resistance of soybean to A. glycines, so genotypes of soybean should be screened for resistance to the aphid at multiple temperatures.     

Chlorophyll loss caused by soybean aphid (Hemiptera: Aphididae) feeding on soybean

The soybean aphid, Aphis glycines Matsumura (Hemiptera: Aphididae), is a worldwide pest of soybean, Glycine max (L.) Merr. Studies to find control methods were initiated in 2000 when it was first detected in North America. A. glycines can reduce yields by as much as 50%, and it is the vector of several viral diseases. A. glycines removes phloem sap, which can result in a reduction of chlorophyll content. Quantification of chlorophyll loss caused by A. glycines feeding on soybean is of vital importance. The SPAD-502 chlorophyll meter is a device that has been used to measure chlorophyll loss caused by nonchewing insects. Chlorophyll loss was studied in no-choice tests on the infested and uninfested leaves of a susceptible check (KS4202). The minimum combined number of days and aphids needed to detect significant chlorophyll loss was 30 aphids confined for 10 d. In a similar experiment, seven resistant entries and two susceptible checks were evaluated. There was no significant chlorophyll reduction between infested and uninfested leaves of five of the resistant entries (K1621, K1639, Pioneer 95B97, Dowling, and Jackson). Percentage of loss of chlorophyll in the susceptible checks was approximately 40%; Jackson and Dowling had a significantly lower percentage loss (13 and 16%, respectively) compared with the susceptible checks. The percentages of chlorophyll loss of K1621, K1639, and Pioneer 95B97 were not statistically different from the percentage of loss of Jackson.     

Effects of induced plant resistance on soybean looper (Lepidoptera: Noctuidae) in soybean

Soybean looper, Chrysodeixis includens (Walker), is one of the most destructive pests of soybean in the southern U.S. Soybean looper defoliation exceeding 20% from R3 (pod initiation) to R5 (pod fill) can result in significant yield loss. In addition, soybean looper is highly resistant to many insecticides. An alternative to insecticide control is induced host plant resistance. In this study, a total of four experiments over 2 years were conducted in which three different elicitors of SAR (systemic acquired resistance), Actigard 50WG (acibenzolar-S-methyl), Regalia (extract of Reynoutria sachalinensis), and methyl jasmonate (MeJA), were applied to soybean at different plant stages to determine if these chemicals could induce plant resistance and lower soybean looper fitness. None of the elicitors of SAR significantly affected soybean looper mortality. However, Actigard 50WG, MeJA, and Regalia had adverse effects on developmental time, defoliation, and pupal weight of soybean looper. Induced effects by Regalia on soybean looper were very limited compared to Actigard 50WG and MeJA. A single application of MeJA reduced pupal weight by 6.8% and delayed larval development by 14.3%. Soybean seed production was not affected by application of elicitors. In conclusion, the results suggest that exogenous elicitors applied in the field can trigger plant resistance against herbivores and this low level of host plant resistance may effectively lessen pest pressure by favoring natural enemy population regulation without reducing seed production.     

Field and laboratory evaluations of soybean lines against soybean aphid (Hemiptera: Aphididae)

The soybean aphid, Aphis glycines Matsumura (Hemiptera: Aphididae), is a major pest of soybean, Glycine max (L.). Merr., that significantly reduces yield in northern production areas of North America. Insecticides are widely used to control soybean aphid outbreaks, but efforts are underway to develop host plant resistance as an effective alternative management strategy. Here, previously identified resistant lines were evaluated in laboratory tests against field-collected populations of soybean aphid and in field-plot tests over 2 yr in South Dakota. Six lines previously identified with resistance to soybean aphid--Jackson, Dowling, K1639, Cobb, Palmetto and Sennari--were resistant in this study, but relatively high aphid counts on Tie-feng 8 in field plots contrasted with its previously reported resistance. Bhart-PI 165989 showed resistance in one of two laboratory tests, but it had relatively large aphid infestations in both years of field tests. Intermediate levels of soybean aphid occurred in field plots on lines previously shown to have strong (Sugao Zairai, PI 230977, and D75-10169) or moderate resistance to soybean aphid (G93-9223, Bragg, Braxton, and Tracy-M). Sugao Zairai also failed to have a significant proportion of resistant plants in two laboratory tests against aphids field-collected in 2008, but it was resistant in laboratory tests with aphids collected in 2002, 2005, and 2006. Overall, results showed that lines with Rag (i.e., Jackson) or Rag1 gene (i.e., Dowling) had low aphid numbers, whereas lines with Rag2 (i.e., Sugao Zairai, Sennari) had mixed results. Collectively, responses of soybean aphid populations in laboratory and field tests in 2008 resembled a virulence pattern reported previously for biotype 3 soybean aphids, but virulence in soybean aphid populations was variable and dynamic over years of the study. These results, coupled with previous reports of biotypes virulent to Rag1, suggest that deployment of lines with a single aphid-resistance gene is limited for soybean aphid management, and that deployment strategies relying on multiple resistance genes may be needed to effectively use plant resistance against soybean aphid.     

The soybean aldehyde dehydrogenase (ALDH) protein superfamily

Aldehyde dehydrogenases (ALDHs) are members of NAD(P)(+)-dependent protein superfamily that catalyze the oxidation of a wide range of endogenous and exogenous highly reactive aliphatic and aromatic aldehyde molecules to their corresponding non toxic carboxylic acids. Research evidence has shown that ALDHs represent a promising class of genes to improve growth development, seed storage and environmental stress adaptation in higher plants. The recently completed genome sequences of several plant species have resulted in the identification of a large number of ALDH genes, most of which still need to be functionally characterized. In this paper, we identify members of the ALDH gene superfamily in soybean genome, and provide a unified nomenclature for the entire soybean ALDH gene families. The soybean genome contains 18 unique ALDH sequences encoding members of five ALDH families involved in a wide range of metabolic and molecular detoxification pathways. In addition, we describe the biochemical requirements and cellular metabolic pathways of selected members of ALDHs in soybean responses to environmental stress conditions.     

Enumerative and binomial sequential sampling plans for soybean aphid (Homoptera: Aphididae) in soybean

Since the discovery of the soybean aphid, Aphis glycines Matsumura, in midwestern U.S. soybean, Glycine max L., in 2000, the aphid has become a significant economic pest. Basic information about estimating population density within fields is unknown. Therefore, we developed two sampling plans to efficiently characterize A. glycines densities. Enumerative and binomial sequential plans were developed using 89 data sets collected from 10 commercial fields sampled during 2001-2003. Re-sampling software was used to validate the enumerative plan on whole plant counts, based on Taylor's power law parameters (a = 9.157 and b = 1.543). For research applications, the enumerative plan was modified to provide an actual precision level of 0.10 (SE/mean), which resulted in an average sample number of 310 individual plants. For integrated pest management (IPM) purposes, we developed an enumerative plan with an actual precision of 0.25, which resulted in an average sample number of 38 individual plants. For IPM applications, the binomial plan will likely be more practical. Binomial plans were developed using two tally thresholds at five action thresholds. Final analysis of the operating characteristic curve for each plan indicated that the tally threshold of > or = 40 aphids per plant, and an action threshold of 0.837 (84% of the plants infested) provided the most correct treat (4%) and no-treat (95%) decisions, with very low incorrect treat (0.5%) and no-treat (0.5%) decisions. A tally threshold of > or = 40 aphids per plant and action thresholds of 84% of plants infested is equivalent to a mean density of 250 aphids per plant, a recently recommended economic threshold. Using this threshold, the minimum required sample number for the binomial plan was 11 plants.     

The use of laser capture microdissection to study the infection of Glycine max (soybean) by Heterodera glycines (soybean cyst nematode)

The soybean cyst nematode (Heterodera glycines) is an obligate parasite of soybean (Glycine max). It is the most destructive pathogen of G. max, accounting for approximately 0.46–0.82 billion dollars in crop losses, annually, in the U.S. Part of the infection process involves H. glycines establishing feeding sites (syncytia) that it derives its nourishment from throughout its lifecycle. Microscopic methods (i.e., laser capture microdissection [LCM]) that faithfully dissect out those feeding sites are important improvements to the study of this significant plant pathogen. Our isolation of developing feeding sites during an incompatible or a compatible reaction is providing new ways by which this important plant-pathogen interaction can be studied. We have used these methods to create cDNA libraries, clone genes and perform microarray analyses. Importantly, it is providing insight not only into how the root is responding at the organ level to H. glycines, but also how the syncytium is responding during its maturation into a functional feeding site.Key words: soybean, Glycine max, soybean cyst nematode, SCN, Heterodera glycines, microarray, gene expression, plant pathogen, parasite, laser capture microdissection     

Analysis of expressed receptor-like kinases (RLKs) in soybean

Receptor-like kinases (RLKs) play crucial roles in cellular signal perception and propagation. To study the evolutionary relationships among RLKs in soybean, a large-scale expressed sequence tags (ESTs) survey for RLKs-related sequences was conducted. By doing BLAST analysis using our database and The Gene Index Database, 605 putative RLK genes were identified. Based on the phylogeny of the kinase domain, these soybean RLKs were classified into 58 different small subfamilies. The phylogenetic analysis of RLKs in soybean, rice and Arabidopsis showed that different subfamilies of RLKs had different functions and could have experienced different selective pressures.     

Analysis of expressed receptor-like kinases （RLKs） in soybean

Receptor-like kinases （RLKs） play crucial roles in cellular signal perception and propagation. To study the evolutionary relationships among RLKs in soybean, a large-scale expressed sequence tags （ESTs） survey for RLKs-related sequences was conducted. By doing BLAST analysis using our database and The Gene Index Database, 605 putative RLK genes were identified. Based on the phylogeny of the kinase domain, these soybean RLKs were classified into 58 different small subfamilies. The phylogenetic analysis of RLKs in soybean, rice and Arabidopsis showed that different subfamilies of RLKs had different functions and could have experienced different selective pressures.