Biological control of crown gall of grapevine, rose, and tomato by nonpathogenic Agrobacterium vitis strain VAR03-1

A nonpathogenic strain of Agrobacterium vitis VAR03-1 was tested as a biological control agent for crown gall of grapevine (Vitis vinifera). When roots of grapevine, rose (Rose multiflora), and tomato (Lycopersicon esculentum) were soaked in a cell suspension of antagonists before planting in soil infested with tumorigenic A. vitis, A. rhizogenes, and A. tumefaciens, respectively, treatment with VAR03-1 significantly reduced the number of plants with tumors and disease severity in the three plant species. The inhibitory effects of treatment with VAR03-1 and the nonpathogenic A. rhizogenes strain K84 on crown gall of rose and tomato were almost identical, and the inhibitory effect of VAR03-1 on grapevine was superior to that of K84. Moreover, VAR03-1 greatly controlled crown gall of grapevine due to tumorigenic A. vitis in the field. VAR03-1 established populations averaging 10(6) colony forming units (CFU)/g of root in the rhizosphere of grapevine and persisted on roots for 2 years. VAR03-1 was bacteriocinogenic, producing a halo of inhibition against those three species of Agrobacterium. This is the first report that a nonpathogenic strain, VAR03-1, can effectively control crown gall caused by tumorigenic A. vitis, A. rhizogenes, and A. tumefaciens.     

放射土壤杆菌MI15菌株生物防治葡萄根癌病的研究

 从内蒙古玫瑰香葡萄冠瘿中分离到一株无致病力的土壤杆菌MI15菌株,经鉴定为放射土壤杆菌(Agrobacterium radiobacter),属生物Ⅰ型。在平皿培养产生的土壤杆菌素,能抑制葡萄根癌土壤杆菌生物Ⅲ型菌株的生长,在温室向日葵幼苗和葡萄嫩枝上接种,MI15菌株能显著地抑制多株不同类型葡萄根癌土壤杆菌诱导形成冠瘿瘤。在琼脂糖凝胶电泳图谱上,MI15菌株有一条质粒带,与生物Ⅰ型根癌士壤杆菌C58菌株的质粒情况基本相同。研究还表明该菌株能在葡萄上存活定殖。     

Effect of antibiosis on antagonist dose-plant disease response relationships for the biological control of crown gall of tomato and cherry

ABSTRACT The crown gall pathosystem was used to evaluate a model that describes the dose-response relationship between biological control agents and plant pathogens. The model predicts that this relationship can become asymptotic, such that increased antagonist doses cannot compensate for deficiencies in disease suppression. Wounded roots of tomato (Lycopersicon esculentum) and cherry (Prunus mahaleb) plants were dipped into different concentrations of the biological control organism Agrobacterium radiobacter strain K84 prior to inoculation with the pathogen A. tumefaciens. Pathogen strains sensitive or resistant to the antibiotic agrocin 84 were used, and for tomato experiments, a derivative of A. radiobacter strain K84 that does not produce agrocin 84 also was included as an experimental treatment. As predicted by the dose-response model, the amount of disease suppression per unit of antagonist decreased with increasing antagonist dose and became asymptotic at high antagonist densities. Control of crown gall of tomato was nearly complete with the combination of A. radiobacter K84 and an agrocin 84-sensitive strain of A. tumefaciens. Pathogen resistance to agrocin 84 or lack of agrocin 84 production by A. radiobacter resulted in antagonist dose-crown gall incidence relationships that were apparently asymptotic at levels of control significantly less than 100%. For field-grown cherry, similar dose-response relationships were observed with higher asymptotic levels of disease suppression obtained when trees were inoculated with an agrocin 84-sensitive A. tumefaciens strain compared with an agrocin 84-resistant pathogen strain. The differences among bacterial strain combinations in the magnitude of the asymptote defined by the dose-response relationships suggest that A. radiobacter impacts a smaller proportion of the pathogen population when the activity of agrocin 84 is muted.     

Evidence of Migration and Endophytic Presence of Agrobacterium tumefaciens in Rose Plants

Agrobacterium tumefaciens was isolated from stem tumors of several rose cultivars showing that the bacterium is the causal agent of aerial galls in rose plants. No differences were observed in the characteristics of the Agrobacterium isolates from crown or aerial galls. Stem inoculation of ten rose cultivars showed that all of them were susceptible to A. tumefaciens but differences in the size of the resulting tumors were observed. The movement of A. tumefaciens in rose plants was demonstrated using two wild type strains and two antibiotic resistant mutants. Three months after inoculation, the inoculated strains were recovered in the roots, crown and below and above the inoculation site but low numbers of pathogenic Agrobacterium cells were isolated. New tumors appeared in 5% of the noninoculated wounds. A. tumefaciens was isolated from the stem at different distances from the tumor in naturally infected plants. In symptomless commercial plants, the isolation from the roots, crown and at different stem levels demonstrated the existence of systemic and latent infections in rose. Direct isolation using a nonselective and selective media with or without a previous enrichment step were efficient methods for isolating tumorigenic Agrobacterium from the different parts of rose plants.     

Crown gall of hop caused by Agrobacterium tumefaciens biovar 1 in South Africa

Crown gall of hop caused by Agrobacterium tumefaciens biovar 1 is reported for the first time from South Africa. The causal organism was inhibited in vitro by the agrocin of A. radiobacter strain D286 but not by that of the control strain K84. Nevertheless, control was achieved on hop stems in glasshouse inoculations by both biological control strains.     

Agrobacterium-Mediated Transformation of Fusarium oxysporum: An Efficient Tool for Insertional Mutagenesis and Gene Transfer

ABSTRACT Agrobacterium tumefaciens-mediated transformation (ATMT) has long been used to transfer genes to a wide variety of plants and has also served as an efficient tool for insertional mutagenesis. In this paper, we report the construction of four novel binary vectors for fungal transformation and the optimization of an ATMT protocol for insertional mutagenesis, which permits an efficient genetic manipulation of Fusarium oxysporum and other phytopathogenic fungi to be achieved. Employing the binary vectors, carrying the bacterial hygromycin B phosphotrans-ferase gene (hph) under the control of the Aspergillus nidulans trpC promoter as a selectable marker, led to the production of 300 to 500 hygromycin B resistant transformants per 1 x 10(6) conidia of F. oxysporum, which is at least an order of magnitude higher than that previously accomplished. Transformation efficiency correlated strongly with the duration of cocultivation of fungal spores with Agrobacterium tumefaciens cells and significantly with the number of Agrobacteruium tumefaciens cells present during the cocultivation period (r = 0.996; n = 3; P < 0.01). All transformants tested remained mitotically stable, maintaining their hygromycin B resistance. Growing Agrobacterium tumefaciens cells in the presence of acetosyringone (AS) prior to cocultivation shortened the time required for the formation of transformants but decreased to 53% the percentage of transformants containing a single T-DNA insert per genome. This increased to over 80% when Agrobacterium tumefaciens cells grown in the absence of AS were used. There was no correlation between the average copy number of T-DNA per genome and the colony diameter of the transformants, the period of cocultivation or the quantity of Agrobacterium tumefaciens cells present during cocultivation. To isolate the host sequences flanking the inserted T-DNA, we employed a modified thermal asymmetric interlaced PCR (TAIL-PCR) technique. Utilizing just one arbitrary primer resulted in the successful amplification of desired products in 90% of those transformants analyzed. The insertion event appeared to be a random process with truncation of the inserted T-DNA, ranging from 1 to 14 bp in size, occurring on both the right and left border sequences. Considering the size and design of the vectors described here, coupled with the efficiency and flexibility of this ATMT protocol, it is suggested that ATMT should be regarded as a highly efficient alternative to other DNA transfer procedures in characterizing those genes important for the pathogenicity of F. oxysporum and potentially those of other fungal pathogens.     

Indole-3-acetic Acid-producing bacteria are associated with cranberry stem gall

ABSTRACT Cranberry stem gall is characterized by tumors that girdle stems, thereby killing all distal leaves, flowers, and fruit. Among bacteria isolated from galls, all 11 isolates that were identified as members of the family Enterobacteriaceae caused galls on 50 to 100% of micropropagated cranberry plants that were inoculated. Four of fifteen isolates identified as Pseudomonas spp. caused galls on 10 to 83% of plants inoculated. Twelve of fifteen isolates identified as either Agrobacterium spp. or Rhizobium spp. caused galls on 10 to 50% of plants inoculated, but the galls were smaller than those caused by members of the family Enterobacteriaceae or Pseudomonas spp. There was a positive correlation between the ability of bacteria to produce IAA in vitro and cause galls. In 2002 and 2003, bacteria were isolated from plant and soil samples collected from beds where stem gall had been observed in the past 2 years and beds where stem gall had never been observed. IAA-producing bacteria were common in all samples, although trends were different across years. The results of this study support the hypothesis that IAA-producing bacteria cause cranberry stem gall and suggest that rather than one bacterial species being the cause, multiple strains of bacteria that produce IAA may be responsible for gall formation.     

Large-scale application of biological control of crown gall in Greece1

Crown gall disease of cultivated plants, caused by Agrobacterium tumefaciens, constitutes a serious problem for the fruit tree, rose and grapevine nurseries in Greece. All three biotypes of A. tumefaciens exist in Greece. Biotypes 1 and 2 have a wide host range being responsible for the disease on fruit trees and roses while biotype 3 isolates have a narrow host range infecting grapevine only. All Greek isolates of biotype 1 and all but 3 isolates of biotype 2 were sensitive to biological control with the antagonistic bacterium A. radiobacter strain K84, but the biotype 3 isolates were insensitive to biocontrol. Experiments on the effectiveness of the method in artificial infections of seedlings as well as in naturally infested soils showed that the method is effective and can be applied without any risk of development of forms insensitive to biocontrol. The use of a lyophilized preparation of K84 with skim milk as suspending medium is recommended. The lyophilized antagonistic bacterium retains its activity and the final concentration (10⁶ cfu ml^-) is adequate to protect treated plants from crown gall.     

The effect of solar heating of soil on natural and inoculated agrobacteria

Solarization trials were carried out over 3 years and in two countries to control crown gall disease on fruit trees and eliminate Agrobacterium . In 1992, agrobacteria in naturally infested soils of two Italian nurseries were monitored before and after solarization. Agrobacteria populations decreased by 99% and 92% after the treatment; however, crown gall incidence did not decrease. In 1993 and 1994 solarization was tested in Oregon in fields artificially infested with two marked strains of A. tumefaciens . In sandy loam soil, the target bacteria were eliminated in 4 weeks, while in silty clay soil the populations were markedly reduced after 2 months of treatment. Crown gall incidence on cherry rootstocks transplanted to the field at the end of 1993 was 3.7% in the sandy loam soil control plots, while no tumours were observed on plants from solarized plots. The use of solarization in combination with reduced doses of metham-sodium was also evaluated.     

Détection moléculaire spécifique de la région vir du plasmide pTi d'Agrobacterium tumefaciens dans les sols et plants au Maroc

Le meilleur moyen de contrôle du crown gall ( Agrobacterium tumefaciens ) est la prévention et donc la détection de la bactérie pathogène dans les sols prévus pour les pépinières. Pour cela nous avons essayé une approche moléculaire utilisant la PCR pour caractériser le plasmide pTi par le couple des amorces spécifiques F14/F44 dans les sols de diverses régions marocaines. Cette technique de détection représente un indicateur potentiel d'avertissement sur l'état d'infection des sols et plants par A. tumefaciens qui permet ainsi de déclencher la lutte biologique par le NoGall, produit commercial australien, hébergeant l'antagoniste K₁₀₂₆.     

大豆凝集素基因lec-s转化烟草>增强对烟草花叶病毒的抗性

 将编码大豆凝集素的lec-s基因插入植物表达载体pBI121中,构建植物重组表达质粒pBI121:: lec-s。由根癌土壤杆菌EHA105介导的叶盘法转化烟草,获得了转基因烟草株系。PCR和RT-PCR检测证明lec-s基因已转入烟草植株中。接种烟草花叶病毒（Tobacco mosaic virus,TMV）进行抗病性试验结果表明,转基因烟草叶片上的病斑数显著减少,说明转基因烟草表现出对TMV的抗性。定量RT-PCR检测发现,接种TMV后,抗病防卫基因（PR-1a、GST1、Pal和hsr515）在转基因烟草叶片中显著上调表达。这些结果表明,大豆凝集素基因lec-s转化烟草可对TMV产生抗性,其作用机制可能在于lec-s基因参与了植物的防卫信号通路,诱导了抗病防卫基因在转基因植株体内的表达,增强了植株对TMV的系统抗性。     

编码harpin_（Xoo）蛋白的基因hpa1_（Xoo）转化菊花增强对蚜虫的抗性

将水稻白叶枯病原菌Xanthomonas oryzae pv.oryzae编码harpinXoo蛋白的hpa1Xoo基因构建植物重组表达质粒pCAMBIA3300-hpa1Xoo-bar,由根癌农杆菌Agrobacterium tumefa-ciensLBA4404介导,叶盘法转化寒菊QX-077,获得了草丁膦转基因寒菊株系。经PCR、Southernblot和RT-PCR检测证明hpa1Xoo已整合到寒菊基因组中。在转基因株系的叶片和植株上抗虫性鉴定表明,转基因寒菊显著抑制桃蚜Myzus persicae的繁殖（t〈0.01）。RT-PCR表明转基因植株中hpa1Xoo能够在转录水平正常表达。hpa1Xoo转化寒菊可以正常表达并提高寒菊对蚜虫的抗性。     

马铃薯Y病毒复制酶基因不同位置cDNA区段介导对PVY的不同抗性

为探究靶序列位置对RNA介导的病毒抗性产生的影响,利用聚合酶链式反应(polymerase chain reaction,PCR)技术扩增马铃薯Y病毒(Potato virus Y,PVY)复制酶基因(nuclear inclusion b,NIb)不同位置的cDNA区段,反向插入双元载体pROKII中,构建了发夹RNA(hairpin RNA,hpR-NA)结构的植物表达载体。将构建的植物表达载体采用冻融法转入农杆菌LBA4404,叶盘法转化烟草NC89,获得转基因植株。攻毒试验表明:PVYNIb基因不同位置cDNA区段介导的对PVY的抗性存在显著差异;3′端1/2处和中间位置的序列可介导高水平的病毒抗性,抗性植株的比例在50%以上,而5′端、5′端1/2处和3′端的序列介导的抗性效率较低,抗性植株的比例仅为10%~30%。Northern杂交显示:抗病植株中RNA的积累量明显低于同类型的感病植株,抗性与RNA积累量呈负相关;抗病转基因植株中有siRNA存在,表明病毒抗性是由RNA介导的。     

Behavior of a Virulent Strain Derived from Agrobacterium radiobacter Strain K84 After Spontaneous Ti Plasmid Acquisition

ABSTRACT The behavior of the virulent transconjugant K84N6 derived from Agrobacterium radiobacter strain K84 after spontaneous Ti plasmid transfer in crown gall tissue in a biocontrol experiment was studied and compared with the behavior of the wild-type A. tumefaciens donor of the Ti plasmid. The main difference between the strains was a greatly reduced ability of the transconjugant to catabolize nopaline. Host range, ability to induce tumors in several fruit trees, and stability of the pathogenic determinants in isolates from tumors did not differ between the strains. Nevertheless, in a biocontrol experiment, the transconjugant was not controlled by strain K84 or K1026 in peach x almond hybrids and survived in the plant rhizo-sphere for 9 months with larger population densities than the wild strain. The appearance and persistence in soil of strains harboring a Ti plasmid in the K84 chromosomal background could represent a risk in the medium term, if they show good competitive ability.     

利用RNAi 介导的抗病性获得抗2 种花生病毒的转基因烟草

 分别以花生条纹病毒红安株系(PStV-Hongan)外壳蛋白基因(CP),花生矮化病毒轻型株系(PSV-Mi)和花生黄瓜花叶病毒(CMV-CA)复制酶基因2a为模板,通过PCR 方法分别得到PStV-CP 5′ 端,PSV-Mi 和CMV-CA 2a3′ 端150 bp 的片段,3 种片段混合物为模板经PCR 拼接得到450 bp 的片段,此拼接片段通过Gateway 系统重组至植物表达载体pK7GW1WG2,得到含反向重复拼接片段的植物表达载体pK450。冻融法导入根癌农杆菌(Agrobacterium tumefaciens)菌株GV3101 后,叶盘法转化本生烟(Nicotiana benthamiana),经PCR 检测,获得转基因植株。对T1 代转基因植株分别人工接种3 种病毒,66. 7% 的植株表现对PStV 免疫,9% 的植株表现对CMV-CA 的恢复抗性,全部植株对PSV 感病。siRNA 的Northern blot 结果表明,所有转基因烟草植株都含有病毒特异siRNA,siRNA 含量随接种后时间延长而衰减。     

Inhibitory effect of ACC deaminase‐producing bacteria on crown gall formation in tomato plants infected by Agrobacterium tumefaciens or A. vitis

This study showed that various rhizosphere bacteria producing the enzyme 1‐aminocyclopropane‐1‐carboxylate (ACC) deaminase (ACCD), which can degrade ACC, the immediate precursor of ethylene in plants, and thereby lower plant ethylene levels, can act as promising biocontrol agents of pathogenic strains of Agrobacterium tumefaciens and A. vitis. Soaking the roots of tomato (Solanum lycopersicum) seedlings in a suspension of the ACCD‐producing Pseudomonas putida UW4, Burkholderia phytofirmans PsJN or Azospirillum brasilense Cd1843 transformed by plasmid pRKTACC carrying the ACCD‐encoding gene acdS from UW4, significantly reduced the development of tumours on tomato plants injected 4–5 days later with pathogenic Agrobacterium strains via wounds on the plant stem. The fresh mass of tumours formed by plants pretreated with ACCD‐producing strains was typically four‐ to fivefold less than that of tumours formed on control plants inoculated only with a pathogenic Agrobacterium strain. Simultaneously, the level of ethylene evolution per amount of tumour mass on plants pretreated with ACCD‐producing bacteria decreased four to eight times compared with that from tumours formed on control plants or plants pretreated with bacteria deficient in ACCD production. Moreover, transgenic tomato plants expressing a bacterial ACCD were found to be highly resistant to crown gall formation relative to the parental, non‐transformed tomato plants. The results support the hypothesis that ethylene is a crucial factor in Agrobacterium tumour formation, and that ACCD‐produced rhizosphere bacteria may protect plants infected by pathogenic Agrobacteria from crown gall disease.     

半夏凝集素基因的克隆及转基因烟草对蚜虫的抑制作用

采用同源序列克隆方法,通过设计特异性引物从甘肃西和半夏叶片基因组DNA中克隆到1条1 069 bp的半夏凝集素基因pta,与以同科内植物的mRNA为模板扩增得到的凝集素基因序列的同源性很高,达到98%以上,功能区完整,具有1条信号肽和3个甘露糖结合区,GenBank登录号AY725425。用该基因替换pBI121载体的GUS基因,正向插入35S启动子之下,构建了植物表达载体pBI-pta。通过农杆菌介导法转化烟草,从20株Kan抗性植株中得到PCR检测阳性植株14株,证明pta已整合到烟草基因组中。对随机挑取的7株PCR阳性植株进行了抗蚜虫(Myzus persicae)筛选试验,结果表明:不同株系蚜口密度抑制率在22.5%~89.4%,平均蚜口密度抑制率56.2%。     

放射土壤杆菌HLB-2菌株防治葡萄根癌病的研究

用放射土壤杆菌HLB-2菌株的悬浮液分别预浸接种根癌土壤杆菌的葡萄苗、向日葵苗,和田间自然带菌的葡萄苗,抑制葡萄冠瘿发生的效果分别为85.6％,83.5％和100％。在田间葡萄痛株上割去冠瘿后的患部,涂上HLB.2菌悬浮液,防治效果为73.1～88.6％。