转拟南芥NPR1基因博B的抗病性及农艺性状表现

通过根癌农杆菌介导法将拟南芥（Arabidopsis thaliana）NPR1基因转入广西主栽保持系水稻品种博B,经连续自交至T3代得到4株纯合株系,PCR检测4株G418抗性植株均为阳性。对这4株转基因植株进行了T1、T2代植株抗病试验及对T3代植株进行了农艺性状的考查,T1、T2代分别进行了水稻白叶枯病菌和稻瘟病菌的接种试验,转基因植株表现出对水稻白叶枯病和稻瘟病的抗性增强。T3代大部分主要农艺性状与野生型相比较一致性高。     

酵母pac1基因介导对葡萄B病毒的抗性

为明确广谱性抗病毒基因—酵母pac1基因对葡萄B病毒(Grapevine virus B,GVB)的抗性效果,通过农杆菌介导的遗传转化,将pac1基因导入西方烟37B,对转基因植株进行PCR鉴定及Southern blot分析,通过病毒摩擦接种观察症状以及实时荧光定量RT-PCR检测植株体内病毒含量,并对转基因植株抗病性进行初步鉴定。结果表明,目的基因pac1成功导入并整合至西方烟37B基因组,共获得10个转基因株系。不同株系的T1代烟草中阳性植株比例为16.7%~72.7%,表明目的基因可成功遗传到子代。接种病毒后转基因植株普遍延迟发病,但后期症状与非转基因对照相似,其中仅1个转基因株系B6具有不表现典型症状等抗性反应。接种植株病毒含量检测中,所有转基因植株均检测到病毒存在,但表现为抗病的B6株系中病毒含量显著低于非转基因对照,表明该转基因植株虽不能完全抵抗GVB侵染,但对GVB具有耐病性。     

转葡聚糖酶和防御素基因的番茄植株及其对番茄灰霉病的抗性

用根癌农杆菌Agrobacterium tumesfaciens介导法，将烟草β-1，3-葡聚糖酶Glucanase基因、苜蓿防御素alfAFP基因及二者双价基因导入番茄Micro—Tom中，以期获得抗灰霉病的转基因株系，并比较验证双价基因是否具有协同作用。绿色荧光蛋白基因（GFP）活性检测、PCR和Southem杂交检测表明，外源基因分别以1—4个拷贝整合到番茄基因组中。对T1代植株进行接种鉴定表明，转基因植株对灰霉病侵染的抵抗能力较对照明显增强，各株系的抗病能力与外源基因的表达相关，且葡聚糖酶基因和防御素基因对抗灰霉菌表现一定的协同作用。T1株系田间抗病鉴定结果表明，转基因株发病程度不同，病情指数在19—28之间，抗病性均高于对照。所得到的稳定表达的株系可作为抗病育种材料进一步研究。     

关于CMV-CP基因番茄植株的抗病表现

黄瓜花叶病毒病(CMV)严重危害番茄生产,降低番茄产量和质量。目前,CMV-CP基因转化番茄在国内外获得成功,已得到抗CMV的转基因番茄植株。本试验以带有CMV-CP基因的番茄品种80-2-1为试材,不带CMV-CP基因的80-2-1作对照,参照JeffersonR.A.方法,研究转基因番茄R1～R4代植株对CMV的抗病表现及外源标志基因的表达。转CMV-CP基因的番茄植株对CMV侵染表现显著的抗性,温室中接种的植株无症率为:R1代94%,R2代95.1%,R3代95.4%,R4代95%;大田中自然发病的植株无症率为:R2代65.6%,R3代76.9%。在转基因的番茄植株中,外源基因GUS(β-gluc…     

抗菌蛋白AP1编码基因对马铃薯的转化及其介导的青枯病抗性研究

根据已知马铃薯抗青枯菌蛋白AP1编码基因序列,合成了特异性引物,经PCR扩增、酶切以及连接、转化等分子操作,成功地获得了AP1编码基因植物表达载体pHap1,该载体具有-2E-P35S-12-ap1-结构。以电激法将pHap1导入根癌农杆菌LBA4404,用叶盘法转化马铃薯青枯病感病品种Mira、中-5-1及金冠试管苗,分别获得了卡那霉素(kan)抗性再生苗。经PCR、Southern、Northern检测,表明ap1基因已成功整合到转基因植株染色体上并正确表达。对Mira品种的转基因植株进行了青枯病抗病性鉴定,结果表明,转基因植株的抗病性比非转基因植株对照明显提高,发病或出现萎蔫症状的时间延迟、病情指数降低。     

葡萄病毒B CP基因植物表达载体构建及烟草遗传转化

葡萄病毒B Grapevine virus B(GVB)是葡萄皱木复合病中栓皮病的病原,为开展抗GVB转基因研究,本研究利用RT-PCR技术克隆GVB外壳蛋白(coat protein,CP)基因,与植物表达载体pRI 101-AN连接构建植物表达载体pRI-GVB CP。采用电击转化法将植物表达载体pRI-GVB CP导入农杆菌LBA4404,并利用农杆菌介导的叶盘转化法将外源基因导入西方烟‘37B’。共获得16个烟草再生株系,PCR检测其中3个株系为阳性,阳性株系播种获得的64株T_1代植株中有30株扩增到目的条带,阳性率为46.9%,表明目的基因GVB cp成功导入烟草并可成功遗传到子代。28株T_1代转基因植株接种病毒进行抗病性鉴定,其中有6株对接种病毒GVB具有抗性。     

芽孢杆菌B47菌株对番茄青枯病的防治作用

用不同接种方法测定番茄内生芽孢杆菌B47菌株对番茄青枯病的室内防效，结果表明，接种B47菌17d后再接种茄青枯雷尔氏菌的植株能较好地防治番茄青枯病，防治效果为81．25％；B47菌和茄青枯雷尔氏菌同时接种的植株对该病的防效较低，仅为16．67％。B47菌株与根围链霉菌St103菌株混合施用室内对番茄青枯病的防效为62．52％，田间防效为81．82％。     

应用组织培养技术离体筛选枸杞抗根腐病变异体的研究

 用宁夏枸杞-宁杞1号的髓组织进行离体培养,诱导产生胚性愈伤组织,并用⁶⁰Co-γ射线进行诱变,以枸杞根腐菌尖孢镰刀菌(Fusarium oxysporum Schlecht)菌株产生的培养滤液为选择剂,筛选抗性变异体,在含毒素60%的条件下,一次性获得抗根腐病毒素的愈伤组织,抗性愈伤组织经稳定性分析,其抗性稳定。抗性愈伤组织在含30%的毒素培养基中可分化成再生植株,共获得25株,其叶片经用根腐菌分生孢子液接种鉴定,有6株抗病;再生植株经过脯氨酸、叶绿素含量分析,二者含量都有所提高;经过过氧化物同工酶酶谱分析,抗性再生植株酶谱带数有增加;说明经筛选得到的抗性再生植株是抗根腐病的变异体。     

青枯菌(Pseudomonas solanacearum)在番茄抗、感病品种根部的吸附、侵入和繁殖

采用不同的方法接种和利用扫描、透射电镜观察,研究不同致病力的青枯菌对番茄抗病及感病品种根部的吸附、侵入与繁殖。发现番茄抗病品种与感病品种的植株体内在菌体数量上有明显差异,而与青枯菌对番茄根部的吸附关系不显著。电镜观察发现青枯菌强致病力菌株菌体能以游离的形式存在于番茄感病品种根部的细胞间隙中,并能降解植株细胞壁、破坏原生质膜;青枯菌强致病力菌株菌体在抗病品种根内和青枯菌强致病力菌株在抗病及感病品种根内均被番茄植株细胞壁吸附,并且被细胞壁周围的浓密物质所包围。     

青枯菌(Pseudomonas solanacearum)在番茄抗、感病品种根部的吸附、侵入和繁殖

 采用不同的方法接种和利用扫描、透射电镜观察,研究不同致病力的青枯菌对番茄抗病及感病品种根部的吸附、侵入与繁殖。发现番茄抗病品种与感病品种的植株体内在菌体数量上有明显差异,而与青枯菌对番茄根部的吸附关系不显著。电镜观察发现青枯菌强致病力菌株菌体能以游离的形式存在于番茄感病品种根部的细胞间隙中,并能降解植株细胞壁、破坏原生质膜;青枯菌强致病力菌株菌体在抗病品种根内和青枯菌强致病力菌株在抗病及感病品种根内均被番茄植株细胞壁吸附,并且被细胞壁周围的浓密物质所包围。     

番茄黄萎病抗病基因Ve的AFLP和SSR分子标记

 本研究以番茄抗病品种05046与感病品种051355配制杂交组合,接种鉴定F1代及F2代分离群体的黄萎病发生情况,结果表明,番茄黄萎病属单基因显性遗传。用545对AFLP引物和101对SSR引物对两个亲本、抗感池及F2代分离群体进行AFLP和SSR分析,得到3个与番茄抗黄萎病基因Ve连锁的AFLP标记和1个SSR标记,分别是E66M84-A、E78M84-D、E66M40-A和SSR599,与抗病基因Ve的连锁遗传距离分别为10.3、14.2、30.5 和12.5 cM。     

木霉T97菌株对几种植物病原真菌的拮抗作用机制和温室防治试验

从土壤中分离了一木霉Trichoderma sp.菌株T97.竞争及对峙培养结果表明,木霉T97对豌豆根腐病菌Fusarium solani f.sp.pisi、番茄灰霉病菌Botrytis cinerea、茄子黄萎病菌Verticillium dahliae、黄瓜枯萎病菌Fusarium oxysporum f.sp. cucumerinum、小麦全蚀病菌Gaeumannomyces graminis var. tritici、小麦根腐病菌Bipolaris sorokiniana和立枯丝核病菌Rhizoctonia solani等7种病原菌有较强的生长竞争优势.光学显微观察表明,木霉T97通过缠绕、附着和穿透的方式寄生立枯丝核菌、番茄灰霉病菌和小麦全蚀病菌.受T97作用后,茄子菌核病菌的菌丝尖端肿大、变粗,豌豆根腐病菌和黄瓜枯萎病菌的菌丝出现断裂等溶菌现象.用T97培养物(0.6%(w/w))处理土壤,对茄子黄萎病和菌核病、黄瓜枯萎病和菌核病以及豌豆根腐病的苗期病害防治效果达66%～81%.用T97孢子悬浮液108cfu/mL在花期喷雾保护黄瓜、辣椒和番茄叶面,对灰霉病的防治效果相当于50%速克灵WP 3 000倍液.     

Potato <Emphasis Type="Italic">R1</Emphasis> resistance gene confers resistance against <Emphasis Type="Italic">Phytophthora infestans</Emphasis> in transgenic tomato plants

Tomato is challenged by several pathogens which cause loss of production. One such pathogen is the oomycete Phytophthora infestans which is able to attack all the aerial parts of the plant. Although a wide range of resistance sources are available, genetic control of this disease is not yet successful. Pyramiding R-genes through genetic transformation could be a straightforward way to produce tomato and potato lines carrying durable resistance to P. infestans. In this work the R1 potato gene was transferred into tomato lines. The tomato transgenic lines were analyzed by using q-RT-PCR and progeny segregation to determine the gene copy number. To test the hypothesis that R1 represents a specifically regulated R-gene, transgenic tomato plants were inoculated with P. infestans isolate 88133 and IPO. All the plants containing the R1 gene were resistant to the late blight isolate IPO-0 and susceptible to isolate 88133. These results provide evidence for specific activation of the R1 gene during pathogen challenge. Furthermore, evidence for enhancement of PR-1 gene expression during P. infestans resistance response was obtained.     

Resistance enhancement of transgenic tomato to bacterial pathogens by the heterologous expression of sweet pepper ferredoxin-I protein

ABSTRACT Expression of a foreign gene to enhance plant disease resistance to bacterial pathogens is a favorable strategy. It has been demonstrated that expressing sweet pepper ferredoxin-I protein (PFLP) in transgenic plants can enhance disease resistance to bacterial pathogens that infect leaf tissue. In this study, PFLP was applied to protect tomato (Lycopersicon esculentum cv. cherry Cln1558a) from the root-infecting pathogen, Ralstonia solanacearum. Independent R. solanacearum resistant T(1) lines were selected and bred to produce homozygous T(2) generations. Selected T(2) transgenic lines 24-18-7 and 26-2-1a, which showed high expression levels of PFLP in root tissue, were resistant to disease caused by R. solanacearum. In contrast, the transgenic line 23-17-1b and nontransgenic tomato, which showed low expression levels of PFLP in root tissue, were not resistant to R. solanacearum infection. The expansion of R. solanacearum populations in stem tissue of transgenic tomato line 24-18-7 was limited compared with the nontransgenic tomato Cln1558a. Using a detached leaf assay, transgenic line 24-18-7 was also resistant to maceration caused by E. carotovora subsp. carotovora; however, resistance to E. carotovora subsp. carotovora was less apparent in transgenic lines 26-2-1a and 23-17-1b. These results demonstrate that PFLP is able to enhance disease resistance at different levels to bacterial pathogens in individual tissue of transgenic tomato.     

Sensitivity to a Phytotoxin from Rhizoctonia solani Correlates with Sheath Blight Susceptibility in Rice

ABSTRACT Sheath blight is one of the most important and intractable diseases of rice (Oryza sativa) where limited control has been achieved using traditional approaches. Quantitative inheritance, extraneous traits, and environmental factors confound genetic analysis of host resistance. A method was developed to isolate and utilize a phytotoxin from Rhizoctonia solani to investigate the genetics of sheath blight susceptibility. Infiltration of the toxin preparation into plant leaves induced necrosis in rice, maize, and tomato. Using 17 rice cultivars known to vary in sheath blight resistance, genotypes were identified that were sensitive (tox-S) and insensitive (tox-I) to the toxin, and a correlation (r = 0.66) between toxin sensitivity and disease susceptibility was observed. Given the broad host range of R. solani, genotypes of host species may be both tox-S and tox-I. A total of 154 F(2) progeny from a cross between Cypress (tox-S) and Jasmine 85 (tox-I) segregated in a 9:7 ratio for tox-S/tox-I, indicating an epistatic interaction between two genes controls sensitivity to the toxin in rice. This work provides the means to genetically map toxin sensitivity genes and eliminate susceptible genotypes when developing sheath blight-resistant rice cultivars.     

Soil solarization for the control of verticillium wilt of greenhouse tomato

Verticillium dahliae, the causal agent of Verticillium wilt of tomato, causes serious damage to crops grown in unheated greenhouses. To control this disease, growers are obliged to employ strong soil disinfestants. The possibility of controllingV. dahliae by using soil solarization during the months of June — August was examined. The soil was covered with transparent polyethylene sheets for 10 weeks. The pathogen could not be isolated from the solarized soil, whereas the inoculum level in the nonsolarized soil remained high (1379–1806 propagules/g soil). The yield from the solarized soil was increased by 112.4% in comparison with the control, and no infected plants were observed. The percentage of infected roots was very low (0.3–0.4%) in relation to the nonsolarized soil (66.7–67.1%). From these results it was concluded that solarization can effectively control Verticillium wilt of greenhouse-grown tomato under the summer conditions in Crete.     

Interactions between Trichoderma harzianum and defoliating Verticillium dahliae in resistant and susceptible wild olive clones

Verticillium wilt (VW) in olive is best managed by an integrated disease management strategy, of which use of host resistance is a key element. The widespread occurrence of a highly virulent defoliating (D) Verticillium dahliae pathotype has jeopardized the use of commercial olive cultivars lacking sufficient resistance to this pathogen. However, the combined use of resistant wild olive rootstocks and Trichoderma spp. effective in the biocontrol of VW can improve the management of VW in olive. In vivo interactions between D V. dahliae and Trichoderma harzianum were studied in olive and wild olive plants displaying different degrees of resistance against this pathogen using confocal microscopy. This multitrophic system included wild olive clones Ac‐4 and Ac‐15, olive cv. Picual, and the fungal fluorescent transformants T. harzianum GFP22 and V. dahliae V138I‐YFP, the latter being obtained in this study. In planta observations indicated that V138I‐YFP colonizes the roots and stems of the olive and wild olive genotypes, and that GFP22 grows endophytically within the roots of them all. YFP fluorescence signal quantifications showed that: (i) the degree of root and stem colonization by the pathogen varied depending upon the susceptibility of the tested wild olive genotype, being higher in Ac‐15 than in Ac‐4 plants; and (ii) treatment with T. harzianum GFP22 reduced the extent of pathogen growth in both clones. Moreover, root colonization by strain GFP22 reduced the percentage of pathogen colonies recovered from stems of olive and wild olive plants.     

Accumulation of phytoalexins in susceptible and resistant near-isogenic lines of tomato infected with Verticillium albo-atrum or Fusarium oxysporum f.sp. lycopersici

The time course of accumulation of two phytoalexins, the terpenoid rishitin and the polyacetylene cis-tetradeca-6-ene-1,3-diyne-5,8-diol, was determined in near-isogenic susceptible and resistant tomato lines inoculated with either Verticillium albo-atrum or Fusarium oxysporum f.sp. lycopersici.Cultivars containing the Ve gene for verticillium wilt resistance accumulated phytoalexins at a rate similar to that in susceptible plants following stem inoculation with V. albo-atrum. Higher amounts of phytoalexins were isolated from susceptible than from resistant plants at 11 days after inoculation. Inoculum concentrations of 10⁵, 10⁶, 10⁷ and 10⁸ conidia ml⁻¹ had no differential effect on phytoalexin accumulation at 3 days after inoculation. Also, no differences were observed between fungal growth in susceptible and resistant cultivars during that period.A cultivar containing the I-1 gene for fusarium wilt resistance contained more rishitin than did susceptible plants at 2 and 3 days after inoculation with 10⁷ conidia of F. oxysporum f.sp. lycopersici ml⁻¹, but at 7 and 11 days after inoculation more rishitin had accumulated in the susceptible plants.No difference was observed between the rate of accumulation of phytoalexin in stem segments from resistant and susceptible plants inoculated by vacuum-infiltration.To estimate the concentration of phytoalexins in the xylem fluid, sap was expressed from vascular tissue and amounts of phytoalexins were determined in the sap and in the expressed tissue. Less than 5% of the phytoalexins present in stem segments was recovered from the sap, indicating that their concentration in the xylem fluid may be relatively low.The role of phytoalexins in resistance to verticillium and fusarium wilt is discussed.     

Diversity and distribution of Ralstonia solanacearum strains in Guatemala and rare occurrence of tomato fruit infection

Fifty-nine Ralstonia solanacearum isolates from diverse crops and regions were collected and characterized to determine the distribution and diversity of this soilborne pathogen in Guatemala. Three distinct types were present: a phylotype I, sequevar 14 strain, probably originating from Asia, infecting tomatoes and aubergines at moderate elevations; a phylotype II, sequevar 6 strain of American origin causing Moko disease in lowland banana plantations; and a phylotype II, sequevar 1 (race 3 biovar 2) strain causing brown rot on potatoes, Southern wilt of Pelargonium spp. and bacterial wilt of greenhouse tomatoes at high elevations. These data on strain diversity will inform effective regional efforts to breed for wilt resistance. A sensitive enrichment method did not detect the pathogen in fruits from naturally infected commercial tomato plants in Guatemalan fields and greenhouses, although it was detected in 6% of fruits from a wilt-resistant hybrid. Low numbers of R. solanacearum cells were also infrequently detected in fruits from plants artificially inoculated in the growth chamber with either race 3 biovar 2 or a phylotype II tomato strain.