Current status in production and utilization of dihaploids from somatic hybrids between eggplant (Solanum melongena L.) and its wild relatives

The major constrains for practical exploitation of the somatic hybrids between eggplant and its wild relatives have been their sterility and tetraploidy which prevented their incorporation into breeding programs. Here we demonstrate that anther culture was successfully utilized to bring back the ploidy level to the diploid status in tetraploid interspecific hybrids between eggplant and the allied species S. integrifolium and S. aethiopicum gr. gilo. Both the relative species are resistant to Fusarium oxysporum f. sp. melongenae and to some strains of bacterial wilt (Ralstonia solanacearum) which are very destructive diseases of eggplant. Dihaploid androgenetic plants were obtained from the somatic hybrids, from the “double somatic hybrid” obtained by sexual cross of the two somatic hybrids [(eggplant + S. aethiopicum) × (eggplant + S. integrifolium)], and from tetraploid backcrossed plants between the somatic hybrid with S. aethiopicum and eggplant. Phenotypical, molecular, biological and biochemical characterization, and also artificial inoculation with Fusarium oxysporum are consistent with a recombination between the genomes of the species involved in the hybridizations. Dihaploids resistant to Fusarium were successfully backcrossed with eggplant. Besides their utility as potential valuable breeding materials, the introgressed lines obtained may be utilized in genetic and molecular studies about the resistance to Fusarium from S. integrifolium and S. aethiopicum gr. gilo.     

嫁接西瓜对西瓜枯萎病菌侵染的防御机制研究

为了揭示嫁接提高西瓜抗枯萎病的机制,该研究以嫁接西瓜为材料,采用扫描电镜观察了枯萎病菌侵染下寄主的组织结构变化,荧光定量分析了相关防卫基因的表达,比较了嫁接西瓜对枯萎病菌侵染的抗感反应。结果显示:(1)枯萎病菌侵染后,与自根西瓜相比,嫁接西瓜的根部木质部导管通过快速形成膜状物、侵填体及细胞壁增厚阻塞菌丝入侵;自根西瓜防御反应较嫁接西瓜晚,严重侵染时薄壁细胞降解,导管组织脱落导致维管系统空洞,从而使植株呈现萎蔫症状,该现象在嫁接西瓜中没有发现。(2)枯萎病菌侵染后,嫁接西瓜比自根西瓜具有较高的防卫基因表达水平,其中:嫁接西瓜中,CHI、APX和PPO基因的表达随枯萎病菌侵染时间的延长而升高,而PAL呈现先升高后降低的表达趋势,但仍高于本底表达;自根西瓜中,仅PPO基因在枯萎病菌侵染后表达上调,而其他基因的表达则是先升高后降低,与嫁接西瓜中的PAL基因表达一致。研究表明,嫁接植株一方面通过快速的组织结构响应,另一方面从转录水平提高了相关防卫基因的表达,最终使植株具有抗病性;推测防御基因在嫁接植株与枯萎病菌互作中的强烈诱导响应可能是嫁接植株抗病的分子机制之一。     

拮抗棉花枯萎病菌的地黄内生细菌筛选、鉴定和促生潜能

[目的]为了筛选棉花枯萎病菌拮抗性菌株资源,从药用植物地黄根块中分离内生细菌,分析优良菌株的促植物生长特性和耐盐碱特性,发掘优良菌株资源,为研发棉花枯萎病生防菌剂提供参考价值.[方法]采用平板对峙法对分离内生细菌进行棉花枯萎病菌拮抗性试验,荧光显微镜观察法研究病原菌菌丝的变化、分光光度计法测定吲哚乙酸(IAA)含量和1...     

棉花抗枯萎病相关ERF-B3亚组转录因子的克隆与表达

以拟南芥ERF-B3亚组转录因子的AP2/ERF结构域为探针,利用NCBI中的棉花(Gossypium hirsutum)EST数据库,通过电子克隆结合RT-PCR方法,从枯萎病菌诱导后的高抗枯萎病品种‘中棉所12’克隆到1个与抗枯萎病相关的ERF-B3亚组转录因子基因GhB301。序列分析结果显示,GhB301基因cDNA全长593 bp,开放阅读框384 bp,编码127个氨基酸,含有一个保守的AP2/ERF结构域。实时荧光定量PCR检测该基因的表达显示,枯萎病菌诱导后,GhB301基因在棉花根中、抗病品种中优势表达;在乙烯、水杨酸、干旱、低温及高盐的诱导下表达量均发生变化。研究结果表明,GhB301基因可能参与了棉花对病原菌、激素及非生物胁迫的应答反应。     

大豆根瘤内抗棉花枯萎病菌株的筛选及其防病试验

【目的】采用优良抗病性内生菌资源来控制棉花枯萎病是一种有效的措施。本研究从大豆根瘤中筛选棉花枯萎病拮抗性内生细菌,探索其对棉花枯萎病菌丝的抑制作用和代表菌株特性,为发掘和应用防病、抗逆优良菌株提供理论基础。【方法】采用对峙法和代谢液培养法对大豆根瘤内生细菌进行棉花枯萎病菌抑菌性筛选,显微观察法研究筛选菌株引起病原菌菌丝变化,通过菌株培养特征、理化特性和16S r DNA序列同源性分析确定菌株系统发育地位,比色法测定DD174耐盐碱性,盆栽试验验证防病效果。【结果】经复筛和代谢液试验有5株拮抗性较强菌株,被作用病原菌菌丝畸形、细胞壁消失、自溶,菌丝基部加粗、分支增多,呈树根状;菌丝被菌苔包埋而溶解、断裂,菌丝末端球形膨大。对棉花枯萎病菌的抑制作用主要通过菌体产生胞外代谢物发挥作用。菌株DD174、DD176和DD179最相似菌株分别为Bacillus oceanisediminis H2T(GQ292772)和B.thuringiensis ATCC 10792T(AF290545),菌株DD165和DD166最相似菌株均为Stenotrophomonas maltophilia LMG 958T(X95923)。DD174能耐受6%盐浓度,p H 10生长良好,具有一定耐盐碱能力。DD174处理组防治效果达76.32%,其他防效均在62%以上,可作为棉花枯萎病的生防菌株资源。【结论】大豆根瘤内存在棉花枯萎病内生拮抗细菌,其中有些菌株具有一定耐盐碱能力,对棉花枯萎病病原菌及病害有一定抑菌和防病作用。     

Combined application of botanical formulations and biocontrol agents for the management of Fusarium oxysporum f. sp. cubense (Foc) causing Fusarium wilt in banana

Plant products along with biocontrol agents were tested against Fusarium wilt of banana caused by Fusarium oxysporum f. sp. cubense (Foc). Of the 22 plant species tested, the leaf extract of Datura metel (10%) showed complete inhibition of the mycelial growth of Foc. Two botanical fungicides, Wanis 20 EC and Damet 50 EC along with selected PGPR strains with known biocontrol activity, Pseudomonas fluorescens 1, Pf1 and Bacillus subtilis, TRC 54 were tested individually and in combination for the management of Fusarium wilt under greenhouse and field conditions. Combined application of botanical formulation and biocontrol agents (Wanis 20 EC + Pf1 + TRC 54) reduced the wilt incidence significantly under greenhouse (64%) and field conditions (75%). Reduction in disease incidence was positively correlated with the induction of defense-related enzymes peroxidase (PO) and polyphenol oxidase (PPO). Three antifungal compounds (two glycosides and one ester) in D. metel were separated and identified using TLC, RP-HPLC (Reverse Phase-High Pressure Liquid Chromatography) and mass spectrometry. In this study it is clear that combined application of botanical formulations and biocontrol agents can be very effective in the management of Fusarium wilt of banana.     

Differential induction of phenylalanine ammonia-lyase activity in date palm roots in response to inoculation with Fusarium oxysporum f. sp. albedinis and to elicitation with fungal wall elicitor

The inoculation of the roots of resistant (BSTN) and susceptible (JHL) cultivars of date palm seedlings byFusarium oxysporum f. sp.albedinis (Foa) induces an increase in activity of phenylalanine ammonia-lyase (E.C. 4. 3. 1. 5., PAL). The post-infectional response in the PAL activity in the resistant cultivar roots was faster and higher than that in the susceptible cultivar. However, the elicitation of the seedlings by the hyphal wall preparation (HWP) ofFoa induces an identical PAL response in the resistant and the susceptible cultivars. The elicitor activity of HWP was dose-dependent, the optimal concentration which induces a maximum PAL activity was 10 mg of mycelium per mL. The elicitor present in the HWP was thermostable since its elicitor activity was maintained after heat treatment (121 °C for 45 min). The treatment of the HWP with protease (Pronase E) does not have an effect on the HWP elicitor activity. However, the treatment of the HWP with sodium periodate inhibits its elicitor activity. This data suggests that the HWP elicitor is a carbohydrate compound. In addition, the HWP elicitor is non-specific since it induces identical responses of the PAL activity in two cultivars showing different behaviors to the pathogen. The absence of specificity of HWP elicitors and the differential response of the PAL activity to the infection byFoa and to the elicitation by the HWP are discussed. An explanation of the general interactions between plant and parasite is proposed.     

RNA-Seq揭示Foc4在外源氧化胁迫(H_2O_2)下的基因表达及细胞代谢变化

尖孢镰刀菌古巴专化型4号小种(Fusarium oxysporum f. sp. cubense race 4,Foc4)是香蕉枯萎病的强致病性病原菌。Foc4在侵染香蕉植株早期必须面对寄主的活性氧迸发。【目的】了解Foc4应对外源氧化胁迫的分子机制。【方法】利用Illumina 2500 RNA-Seq测序平台分析了经外源氧化胁迫(H_2O_2)处理的Foc4与对照在转录组水平的基因表达差异。【结果】在外源氧化胁迫条件下,Foc4的生长受到抑制。转录组测序获得了超过2千万条clean reads。进一步的差异基因表达分析以差异倍数FC (fold change)≥2且FDA值≤0.001为选择标准,发现496个基因表达上调,298个基因表达下调。GO功能富集分析显示,429个基因比对到GO功能分析数据库,在这些差异表达基因中,许多与代谢过程、生物调节、细胞过程和刺激应答有关。KEGG通路富集分析显示,有141个表达差异显著基因比对到KEGG中的50条代谢途径。其中,主要是各类氨基酸代谢途径、脂肪酸代谢途径。同时也包括与抗氧化胁迫直接相关的代谢途径,包括DNA的损伤修复、类胡萝卜素的生物合成、过氧化物酶体、谷胱甘肽代谢等。【结论】这些结果暗示,为了在强氧化胁迫环境下生存,Foc4细胞从包括直接应对氧化胁迫的信号调控途径在内的物质代谢和能量代谢均发生改变以应对环境变化的胁迫。     

棉花抗枯萎病相关转录因子基因的克隆与表达

以枯萎病菌诱导棉花基因表达谱中获得的差异表达bZIP作为探针,采用电子克隆结合RT-PCR方法从棉花抗枯萎病品种‘中棉所12’中克隆了1个TGA转录因子基因,命名为GhTGA2.2。序列分析表明,该基因的cDNA全长1 356bp,编码451个氨基酸,预测分子量为50.04kD,等电点为5.85,含有保守的bZIP结构域。系统进化树分析表明,GhTGA2.2属于bZIP亚家族的TGA转录因子,与拟南芥AtTGA2、烟草NtTGA2.2亲缘关系最近。qRT-PCR分析表明,经枯萎病菌诱导后,GhTGA2.2基因在抗病品种中呈上调表达,随处理后时间的推移,其相对表达量呈先升高后降低的趋势,并于处理后24h表达量达到最大;水杨酸诱导后1h,GhTGA2.2基因相对表达量迅速增加;茉莉酸和乙烯诱导后GhTGA2.2基因的相对表达量明显降低,呈下调表达。研究推测,GhTGA2.2基因可能通过水杨酸信号传导途径参与对枯萎病菌的防御反应。     

18份广东香蕉种质对枯萎病的抗性评价

【背景】香蕉枯萎病是世界性的香蕉毁灭性病害,尚无有效药剂防控,筛选抗病品种是目前理想的防治方法。【方法】采用组培苗伤根接种法,研究了18份香蕉种质对香蕉枯萎病菌4号生理小种的抗性水平,并根据病情指数进行抗性分级。【结果】在供试的18份香蕉种质中,2份(东莞大蕉、抗枯5号)高抗,2份(碧盛、大丰)抗病,3份(抗枯1号、粉杂、农科1号)中抗,7份(粤优抗1号、广东-741、泰国B9、大蕉、台湾8号、海贡蕉、威廉斯8818)感病,4份(巴西、广东2号、广粉1号、粉蕉)高感。【结论与意义】不同香蕉种质对香蕉枯萎病菌4号生理小种的抗病性存在较大差异,本研究初步筛选出7份抗枯萎病的香蕉种质,为香蕉枯萎病抗病育种提供了依据,为病区种植香蕉品种提供了有效参考。     

棉花EIN3/EIL家族基因序列分析及GhEIL3基因克隆

该研究以拟南芥AtEIN3基因为探针,从陆地棉TM-1全基因组测序数据库中筛选其同源序列,序列分析得到16条具有EIN3结构域的基因序列。对陆地棉EIN3/EIL家族基因的基因结构、系统进化、序列相似性及结构域分布情况进行分析,发现它们与拟南芥EIN3/EIL家族基因在N端具有较高相似度,其中15条基因序列包含5个保守结构,1条包含4个保守结构。在此基础上,采用RT-PCR方法从抗枯萎病的陆地棉品种‘中棉所12’中克隆得到一个新的EIN3/EIL家族基因,命名为GhEIL3(GenBank登录号为KY072936)。序列分析表明:GhEIL3基因开放阅读框长1 092bp,编码363个氨基酸,含有一个EIN3结构域。实时荧光定量PCR(qRT-PCR)结果显示,GhEIL3基因在枯萎病菌诱导后呈上调表达,诱导后1h其相对表达量达到最大值,推测GhEIL3基因可能参与棉花对枯萎病菌的防御反应。     

番茄中Rpi-blb2同源基因的挖掘与鉴定

番茄晚疫病是番茄生产中的主要病害之一,经常会造成较大的经济损失。晚疫病生理小种的变异和进化常会导致番茄品种原有的遗传抗性丧失,因此不断挖掘新的抗性基因,改良番茄晚疫病抗性是番茄抗病育种的长期任务。该研究采用BLAST同源比对的方法,以马铃薯野生近缘种的晚疫病抗性蛋白序列Rpi-blb2为种子序列,在NCBI蛋白质序列数据库中检索得到11条番茄蛋白质序列,这些序列与种子序列相似性为78%~83%,属于番茄疾病抗性蛋白家族,并对该家族成员进行了基因结构、基因定位、序列保守结构域和进化关系等分析。结果表明:该家族中10条序列分布在第Ⅵ条染色体上,1条分布在第Ⅴ染色体上;6号染色体上的10序列呈现2个抗病基因簇分布,在染色体上分别占据2个和3个基因位点;10条同源蛋白是Rpi-blb2的共同垂直同源蛋白,但不具有平行同源关系,大多数成员定位于细胞质。按照蛋白质保守结构域和基因定位的不同可分为三类,第一类共4条系列,包含有DUF3542和NB-ARC两个保守结构域特征序列;第二类共6条序列,与马铃薯Rpi-blb2蛋白一样,仅包含NB-ARC保守结构域特征序列,在这2类蛋白序列的NB-ARC结构域均位于序列中部;第三类(仅包含XP_004239406.1)虽然也具有与第一类蛋白相似的DUF3542和NB-ARC结构域,但在结构域两端的非保守区序列较短,且位于5号染色体上,因此将其单独归为1类。前两类蛋白成员相应的基因具有1~2个内含子,第3类蛋白不含内含子。该研究结果为利用生物技术选育番茄抗性品种提供了理论基础。     

Impact of Crown Rust (Puccinia coronata f. sp. avenae) on Competitive Interactions between Wild Oats (Avena fatua) and Stipa (Nassella pulchra)

Crown rust of oats (Puccinia coronata f. sp. avenae) was investigated as a biocontrol agent for wild oats (Avena fatua) on San Clemente Island, California. Successful restoration of the native habitats of this island will involve the reduction of wild oats and revegetation with native grasses such as stipa (Nassella pulchra). Determination of the outcome of interference between wild oats and stipa is important in the prediction of the success of the biocontrol agent. An addition series design was used to investigate these interactions, with densities of each species ranging from 0 to 2000 seeds per m². Eight replicates were established, four of which were randomly chosen and infected with the pathogen. After 75 days, plant height, dry weight, and seed production were measured. The data were fit to a hyperbola surface model by use of a nonlinear regression procedure. Results indicate that wild oat is the superior competitor at the seedling stage; however, early rust infection greatly reduces fitness of wild oats, causing an increased fitness for stipa. Simulations with a plant community model constructed from the results of the greenhouse experiment and information in the literature indicated that an equilibrium may be established between wild oat and stipa if high initial seeding rates of stipa are used in revegetation.     

Bullera begoniae sp. nov. and Bullera setariae sp. nov., two new species of ballistoconidium-forming yeasts in the Bullera variabilis (Bulleribasidium) cluster isolated from plants in Taiwan

Two strains of xylose-containing and Q-10-having ballistoconidiogenous yeasts isolated from plant leaves collected in Taiwan were found to represent two new species of the genus Bullera. In the phylogenetic trees based on the sequence analysis of 18S rDNA and D1/D2 domain of 26S rDNA, these species are located in the Bullera variabilis (Bulleribasidum) cluster in Hymenomycetes. They are described as Bullera begoniae sp. nov. and Bullera setariae sp. nov., respectively.     

Isolation of Endophytic Actinomycetes From Roots and Leaves of Banana (Musa Acuminata) Plants and Their Activities Against Fusarium oxysporumf. sp. cubense

Two hundred and forty-two actinomycete strains were isolated from the interior of leaves and roots of healthy and wilting banana plants. Most of them were streptomycetes, Streptomyces griseorubiginosus-like strains were the most frequently isolated strains. Community analysis demonstrated increased actinomycete diversity in wilting leaves compared to that in healthy leaves, similar actinomycete communities were found in wilting and healthy roots. Screening of the isolates for antagonistic activity against Fusarium oxysporumf. sp. cubenserevealed that the proportion of antagonistic streptomycetes in healthy roots was higher than that in wilting roots (P < 0.01), but no difference was found between antagonistic strains isolated from healthy and wilting leaves. The potential biological control of Panama disease of banana by endophytic streptomycetes, especially Streptomyces griseorubiginosus-like strains was discussed.     

Sequence of the E. coli O104 antigen gene cluster and identification of O104 specific genes

The Escherichia coli O104 polysaccharide is an important antigen, which contains sialic acid and is often associated with EHEC clones. Sialic acid is a component of many animal tissues, and its presence in bacterial polysaccharides may contribute to bacterial pathogenicity. We sequenced the genes responsible for O104 antigen synthesis and have found genes which from their sequences are identified as an O antigen polymerase gene, an O antigen flippase gene, three CMP-sialic acid synthesis genes, and three potential glycosyl transferase genes. The E. coli K9 group IB capsular antigen has the same structure as the O104 O antigen, and we find using gene by gene PCR that the K9 gene cluster is essentially the same as that for O104. It appears that the distinction between presence as group IB capsule or O antigen for this structure does not involve any difference in genes present in the O antigen gene cluster. By PCR testing against representative strains for the 166 E. coli O antigens and some randomly selected Gram-negative bacteria, we identified three O antigen genes which are highly specific to O104/K9. This work provides the basis for a sensitive test for rapid detection of O104 E. coli. This is important both for decisions on patient care as early treatment may reduce the risk of life-threatening complications and for a faster response in control of food borne outbreaks.     

In vitro efficacy of <Emphasis Type="Italic">Hyptis suaveolens</Emphasis> L. (Poit.) essential oil on growth and morphogenesis of <Emphasis Type="Italic">Fusarium oxysporum</Emphasis> f.sp. <Emphasis Type="Italic">gladioli</Emphasis> (Massey) Snyder &; Hansen

Hyptis suaveolens L. (Poit.) essential oil was tested in vitro on the growth and morphogenesis of Fusarium oxysporum f.sp. gladioli (Massey) Snyder & Hansen, which causes Fusarium corm rot and yellows in various susceptible cultivars of gladiolus. The fungitoxicity of the oil was measured by percentage radial growth inhibition using the poisoned food technique (PF) and volatile activity assay (VA). The mycelial growth of the test fungus was completely inhibited at 0.998 and 0.748 μg ml⁻¹ concentration of oil in PF and VA, respectively. Essential oil was found to be fungicidal in nature at 1.247 and 0.998 μg ml⁻¹ concentration of oil in PF and VA, respectively. Determination of conidial germination in the presence of oil was also carried out and it was found that the oil exhibited 100% inhibition of conidial germination at 0.450 μg ml⁻¹ concentration. The effect of essential oil on the yield of mycelial weight was observed and it was found that at 0.873 μg ml⁻¹ concentration no mycelium was recorded and 100% inhibition was observed. The fungitoxicity of oil did not change even on exposure to 100°C temperature or to autoclaving, and the oil also retained its fungicidal nature even after storage of 24 months. The main changes observed under light microscopy after oil treatment were a decrease and loss of conidiation and anomalies in the hyphae such as a decrease in the diameter of hyphae and granulation of cytoplasm. The treatment of the oil also showed highly reduced cytoplasm in the hyphae, showing clear retraction of the cytoplasm from the hyphae and ultimately in some areas hyphae without cytoplasm were also found. GC-MS studies of the essential oil revealed that the oil consisted of 24 compounds with 1,8-cineole as major component accounting for 44.4% of the total constituents.     

Epistemma neuerburgii (Apocynaceae, Periplocoideae): A new epiphytic species from Nyungwe National Park, Rwanda, Africa

A new species, Epistemma neuerburgii is described. This species was discovered in the Nyungwe National Park, Rwanda, Tropical Africa during a field investigation of epiphytes in montane rainforest. It was, however, also found in Uganda, in similar environment. E. neuerburgii is the fourth species described for Epistemma, but the other three species are found about 2000 km to the west in Cameroon, Gabon, Ghana, Ivory Coast and Nigeria. E. neuerburgii is an epiphytic climber distinguished by fairly large lanceolate leaves and conspicuous reddish-purple flowers with corolline white, densely hairy corona lobes.     

SCAR,RAPD and RFLP markers linked to a dominant gene (Are) conferring resistance to anthracnose in common bean

Anthracnose, caused by the fungusColletotrichum lindemuthianum, is a severe disease of common bean (Phaseolus vulgaris L.) controlled, in Europe, by a single dominant gene,Are. Four pairs of near-isogenic lines (NILs) were constructed, in which theAre gene was introgressed into different genetic backgrounds. These pairs of NILs were used to search for DNA markers linked to the resistance gene. Nine molecular markers, five RAPDs and four RFLPs, were found to discriminate between the resistant and the susceptible members of these NILs. A backcross progeny of 120 individuals was analysed to map these markers in relation to theAre locus. Five out of the nine markers were shown to be linked to theAre gene within a distance of 12.0 cM. The most tightly linked, a RAPD marker, was used to generate a pair of primers that specifically amplify this RAPD (sequence characterized amplified region, SCAR).The research was supported by the CNRS and the Ministère Français de l'Education Nationale