苹果根癌病菌系及生物防治的初步研究

 从山东莱阳苹果园的苹果冠瘿瘤中分离到18株根癌土壤杆菌。经过鉴定有3株是Agrobacterium tumefaciens(原生物1型),其余15株是A.rhizogenes(原生物2型)。这些菌株所诱导的冠瘿瘤中均能合成胭脂碱(nopaline),属于胭脂碱型Ti质粒的根癌土壤杆菌。通过室内平皿检测发现,分离到的菌株均对K84菌株所产生的土壤杆菌素敏感。在温室指示植物向日葵幼苗上K84和K1026均能完全抑制冠瘿瘤的形成。     

广宿主Ti质粒virA毒力基因对窄宿主根癌农杆菌MI3—2菌株宿主范围的扩展作用

生物3型葡萄根癌农杆菌MI3—2有很窄的宿主范围,除了葡萄外,只对少数植物能致瘤。我们先前的研究曾观察到,MI3—2菌株的DNA与广宿主根癌农杆菌A348的PTiA6质粒毒力区virA基因座和T—DNA的tms基因不同源。本实验把含有PTiA6质粒T_L—DNA片段及毒力区各基因座(virA,B,G,c,D和E)片段的粘粒(cosmid),通过三菌接合引入MI3—2菌株,组成局部二倍体,使之与MI3—2Ti质粒中相对应的基因互补。用这种转移接合子感染植物表明,引入有T_L—DNA片段或virA基因座的MI3—2,均能使宿主范围有所扩大,并能在落地生根致瘤。本文提出了广宿主virA基因对根癌农杆菌宿主范围的作用。     

广宿主Ti质粒virA毒力基因对窄宿主根癌农杆菌MI3—2菌株宿主范围的扩展作用

 生物3型葡萄根癌农杆菌MI3-2有很窄的宿主范围,除了葡萄外,只对少数植物能致瘤。我们先前的研究曾观察到,MI3-2菌株的DNA与广宿主根癌农杆菌A348的PTiA6质粒毒力区virA基因座和T-DNA的tms基因不同源⁽¹⁾。本实验把含有PTiA6质粒T_L-DNA片段及毒力区各基因座(virA,B,G,c,D和E)片段的粘粒(cosmid),通过三菌接合引入MI3-2菌株,组成局部二倍体,使之与MI3-2Ti质粒中相对应的基因互补。用这种转移接合子感染植物表明,引入有T_L-DNA片段或virA基因座的MI3-2,均能使宿主范围有所扩大,并能在落地生根致瘤。本文提出了广宿主virA基因对根癌农杆菌宿主范围的作用。     

抗根癌菌剂防治核果类果树根癌病的研究

本文利用引进的放射土壤杆菌K84菌株对我国果树根癌病进行了防治效果的研究，主要包括室内、温室和田间抑菌效果的检验、生产用培养基的改进和菌剂剂型的选择等。室内和温室试验表明，K84菌株能够抑制所有供试的生物Ⅱ型和部分生物Ⅰ型的根癌土壤杆菌，而对来自葡萄的Ⅲ型病菌无效。桃树的田间试验表明K84菌株对人工接种的病菌预防效果可达100％，对自然重茬土壤内的根癌菌也有明显的防治效果，苗龄越小、处理越早，防治效果越好。草炭制剂明显优于液体制剂，其保存期长而且易于运输。本文还介绍一种适于大规模生产的培养基WY。     

葡萄根癌土壤杆菌的不同生化型对葡萄及其它植物致病性的初步研究

 从北京、内蒙古、吉林、辽宁、山东等地采到的葡萄根癌病标本中,分离到根癌土壤杆菌[Agrobacterium fumefaciens(Smith&Town.) Conn]3种生化型菌株。它们对玫瑰香葡萄的致病性明显不同。生化Ⅲ型菌株致病性强,形成大、中型瘤。生化Ⅰ型、Ⅱ型菌株致病性弱,只形成小瘤。生化Ⅲ型的不同菌株对葡萄、向日葵、烟草、番茄等8种植物的致病性也很不同。一类只能侵染葡萄,是窄寄主型;另一类除葡萄外,还能侵染向日葵、烟草、番茄,是宽寄主型。玫瑰香、龙眼等葡萄对3种生化型菌株比较敏感;莎巴珍珠、贝达等比较抗病。生化Ⅲ型和Ⅰ型的一些菌株也能侵染梨、李、杏、樱桃、榆叶梅等几种果树。从山东白雅葡萄上分离的BS33-10菌株(胭脂碱质粒类型)接种在烟草、番茄上能形成畸嵌瘤。     

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％。     

我国部分地区土壤杆菌的数值分类及DNA同源性分析

 本研究对我国42株土壤杆菌待测菌株和28株参比菌进行了数值分类和DNA同源性分析,结果将41株土壤杆菌与2株根瘤菌分在3个土壤杆菌种内,其中23株来自内蒙等地葡萄的菌株分在葡萄土壤杆菌种内,10株来自山东等地樱桃、桃树的菌株为发根土壤杆菌,8株来自4个地区4种植物的菌株及分自宁夏刺果甘草的2株根瘤菌菌株参比菌分在根癌土壤杆菌种内。1株土壤杆菌和1株根瘤菌的分类地位有待进一步研究。     

泰安樱桃根癌病致病菌分离及生物型鉴定

由致病性土壤杆菌侵染引起的根癌病严重影响了樱桃树的生长?利用选择性培养基, 以阿糖醇和赤藓糖醇作为不同碳源, 从山东泰安食用樱桃根部瘤状组织和感病根际土壤中分离到土壤杆菌100余株, 通过胡萝卜切片法和番茄茎部针刺接种法从中筛选到31株有致瘤活性的菌株?经16S rDNA和recA基因序列分析, 结合生物型检测鉴定出96%以上的致病菌属原生物Ⅱ型(Agrobacterium rhizogenes), 只有1株致病菌为原生物Ⅰ型(A. tumefaciens)?根据Ti质粒上致病相关基因保守序列设计3对引物, 12株有致瘤活性菌株中11株PCR扩增到目的条带?经分子生物学测定和高压纸电泳检测, 所有致病菌株的质粒均为胭脂碱型?建立了致病型土壤杆菌的分子生物学快速鉴定方法, 为定向选择生防菌株提供基础?     

宁波樱花根癌病病原细菌鉴定

从浙江宁波14个樱花（Cerasus Mill.）病株样本上分离到多株土壤农杆菌菌株,其中10个菌株可致指示寄主番茄形成肿瘤,对菌株Czx-3的16S rDNA序列比对,确定樱花根癌病是由根癌土壤杆菌（Agrobacterium tumefaciens）侵染所致。进一步开展的标准生化和生理反应测试、碳源利用和选择性培养基等试验,鉴定出7个根癌病菌株为土壤农杆菌（A. tumefaciens）生物型2型,占70%,为优势种群,其余3个菌株为生物型1型。对樱花根癌病菌分类地位的确定,樱花种苗的检疫和病害控制奠定了理论基础。     

土壤杆菌K1026对果树根癌病的生物防治（英文）

由致病性根癌土壤杆菌Agrobacterium spp.侵染引起的根癌病对山东及我国其他省份的樱桃、桃及其他果树生产造成严重的影响。土壤杆菌Agrobacterium rhizogenes K1026在澳大利亚已制成生防菌剂并在全球多个国家实现商品化,但在我国果树育苗和生产行业中还未得到广泛应用。本文从山东樱桃和桃树根癌组织及感病土壤中分别分离到100余株致病性土壤杆菌,经鉴定均属于生物Ⅱ型土壤杆菌;利用双层平板抑菌试验,检测了37株病原菌对菌株K1026的敏感性,结果显示,所有菌株均对K1026产生的抗生物质敏感,可以在Stonier’s培养基上产生5种以上不同类型的抑菌圈;胡萝卜切片试验显示,菌株K1026可完全抑制37株病原菌对胡萝卜的致瘤活性。上述结果为土壤杆菌K1026用于山东省樱桃及桃树根癌病的生物防治奠定了基础。     

Chromosomal and Ti plasmid characterization of tumorigenic strains of three Agrobacterium species isolated from grapevine tumours

Fifty-six tumorigenic Spanish grapevine strains of Agrobacterium spp. were tested for biovar classification, pathogenicity on several hosts, opine utilization, 16S rRNA gene sequencing and PCR amplifications using five primer sets targeting chromosomal and Ti plasmid genes. Fifty of them belonged to A. vitis (biovar 3), three to A. tumefaciens (biovar 1) and three to A. rhizogenes (biovar 2). All strains were tumorigenic on grapevines. Most A. vitis strains were also pathogenic on tomato and tobacco plants, while the three A. tumefaciens strains were only pathogenic on grapevine. Although most A. vitis strains used octopine, 12 utilized neither octopine nor nopaline. 16S rRNA gene sequencing clearly distinguished between strains belonging to the three species. Those of A. vitis could be further divided into three chromosomal backgrounds according to their 16S ribosomal RNA gene sequences. No universal primer pair was found for the detection of all three Agrobacterium species isolated from grapevine. DNA from all A. vitis strains was amplified with the chromosomally-encoded pehA primer pair. In both A. vitis and A. tumefaciens a correlation was observed between the amplifications obtained using the tmr and the virA Ti-plasmid-targeting primer pairs. Three types of Ti plasmid were found in A. vitis strains according to their PCR amplifications and opine utilization profiles. A given chromosomal background harboured only one type of Ti plasmid within the strains from each analysed sample, showing a strong association between chromosomal backgrounds and Ti plasmids in A. vitis .     

K84的生物防治效果与土壤杆菌Ti质粒类型的关系

用K84对我国不同寄主来源的68个Agrobacterium tumefaciens菌株进行生物防治试验,结果表明:K84对我国桃树菌株的防治效果显著,对新疆的啤酒花菌株也有效;但对浙江萧山的部分啤酒花菌株无效;对甜菜菌株的防治效果不佳;对葡萄菌株基本上无效。对致病菌株的质粒类型的研究结果表明:桃树、啤酒花和甜菜菌株基本上都是胭脂碱(Nopaline)型质粒、葡萄菌株基本上是章鱼碱(Octopine)型质拉。试验中还发现不少胭脂碱型菌株不能被K84所防治。此外,在甜菜和毛白杨菌株中也发现了对细菌素A84(agrocin84)敏感,但不能被K84防治的菌株。     

Opine utilization by Spanish isolates of Agrobacterium tumefaciens

Among 115 Spanish isolates of Agrobacterium tumefaciens from 13 hosts and 47 locations, biotypes 1, 2, and 3 were identified. Most isolates were of biotype 2. Biotype 3 isolates were obtained only from grapevine, and all were resistant to agrocin 84. Nopaline was utilized by 84% of isolates. Only three isolates did not utilize either octopine, nopaline or mannopinic acid.
Isolates from fruit trees were mostly biotype 2 and utilized only nopaline; some isolates from a peach x almond hybrid utilized mannopinic acid. Rose and osier isolates were biotype 1 and utilized only nopaline. Most grapevine isolates were biotype 3, but varied widely in opine utilization.
With one exception all isolates sensitive to agrocin 84 catabolized nopaline; no biotype 3 strains catabolized mannopinic acid and no biotype 1 or 2 strains catabolized octopine.     

Results of seven years of biological control of Agrobacterium tumefaciens in Spain1

Biological control of Agrobacterium tumefaciens using the K84 strain of A. radiobacter has been practised in Spain since 1979. Strain K84 is produced in Sevilla by the Agricultural Research Service and is mixed with peat. This formulation is authorized for use on stone fruits and roses, since results of various trials on rootstocks of these crops have shown that K84 is effective in controlling the disease in naturally infected nursery soil, as well as in experimental tests carried out on artificially inoculated soil. Results of biological control trials have shown that K84 has some effect on A. tumefaciens strains resistant to agrocin 84. Factors other than susceptibility to agrocin 84 influence the efficacy of biological control. This was demonstrated by using a K84 agrocin non-producer strain which proved effective against both resistant and susceptible strains. Biological control on apple or pear is not yet authorized in Spain due to a paucity of data on field trials. However, initial pot tests showed good K84 activity on these hosts, against Spanish strains of A. tumefaciens. On grapevine, most of the A. tumefaciens strains that were isolated belong to biotype 3 and are resistant to agrocin 84. Because the disease is systemic on this species the use of K84 is not authorized.     

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.     

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.     

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.     

Inhibition of crown gall formation by Agrobacterium radiobacter biovar 3 strains isolated from grapevine

Graft unions of nursery stock of grapevine (Vitis vinifera L.) collected in Japan yielded pathogenic and nonpathogenic strains of Agrobacterium. On the basis of classical diagnostic tests, a sequence analysis, and a multiplex polymerase chain reaction method previously reported, the pathogenic strain was identified as Agrobacterium tumefaciens biovar 3, whereas the nonpathogenic strains were assigned to Agrobacterium radiobacter biovar 3. Stems of tomato (Lycopersicon esculentum Mill.) seedlings were inoculated with both A. tumefaciens biovar 3 strain G-Ag-27 as a pathogen and one of the control strains isolated from grapevine or A. radiobacter biovar 2 strain K84 as competitors to assay the suppression of gall formation caused by the pathogen. In a test with a 1 : 1 pathogen/nonpathogen cell ratio, all A. radiobacter biovar 3 strains reduced gall incidence and size compared to that of the positive control inoculated only with the pathogen. Strain VAR03-1 was especially effective in reducing the incidence of gall formation on grapevine and reduced gall size by 84%–100% of those on the positive control. Many tested nonpathogenic biovar 3 strains were bacteriocinogenic, causing an inhibition zone against A. tumefaciens biovar 3 strains on YMA medium. Strain VAR03-1 was the most effective against indicator strains and appears to be a promising agent for controlling crown gall of grapevine.     

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.