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.     

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.     

土壤杆菌K1026对果树根癌病的生物防治

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.     

Use of the Genetically Engineered Agrobacterium Strain K1026 for Biological Control of Crown Gall

Strain K84 of Agrobacterium (formerly called A. radiobacter) has been a successful biocontrol agent of crown gall disease for almost 30 years all over the world. In spite of its demonstrated efficiency, the most important risk of failure when using strain K84 is the possibility of transfer of plasmid pAgK84 to pathogenic Agrobacterium strains. pAgK84 codifies production of and immunity to agrocin 84, the main factor involved in crown gall biocontrol by strain K84. Then, a second generation of strain K84 was obtained and the genetically engineered strain was called K1026. It contains a deletion in the transfer region of pAgK84. To date, a considerable number of studies have been performed to compare both strains in its ability to control crown gall, plasmid transfer, antibiotic production, root colonization and survival in the rhizosphere. The aim of this review is to discuss all this comparative available information which advises that strain K1026 should be used as a biopesticide to safeguard biocontrol of crown gall wherever strain K84 is employed.     

Eucalyptus occidentalis plantlets are naturally infected by pathogenic Agrobacterium tumefaciens

In the Sidi M’djahed nursery (Algeria), over 60,000 eucalyptus (Eucalyptus occidentalis) plantlets exhibited tumour-like growths localized at the crown of the plants that resembled crown galls caused by Agrobacterium tumefaciens. Bacteria colonizing the galls were isolated and purified. Most (22 out of 24) of the isolates had cultural and biochemical characteristics similar to those of strains of the biovar 1 of A. tumefaciens. Twenty out of 22 Agrobacterium isolates induced tumour formation on various test plants. In PCR experiments, DNA extracted from these virulent strains yielded an amplification signal corresponding to a 247-bp fragment located within the virulence region of nopaline type Ti plasmid. Consistent with this, the opine nopaline was detected in the tumours induced on test plants – but not on eucalyptus plants. Nopaline was degraded by the 20 pathogenic isolates that were also sensitive to agrocin 84, indicating the presence of a nopaline-type pTi in these strains. The chromosomal region encoding the 16S rRNA was analyzed in a sub-population of the pathogenic agrobacterial isolates. The analyzed strains were found to belong to the ribogroup of the reference strain B6. Interestingly, Eucalyptus camaldulensis and Eucalyptus cladocalyx grown in the same nursery and in the same soil substrate developed no galls.     

Chromosomal and Plasmid Diversity of Agrobacterium Strains Isolated from Ficus benjamina Tumors

Agrobacteria were previously isolated from tumors developing on branches and aerial and hypogeous roots of weeping fig plants in Italy and in The Netherlands. A representative group of 48 strains was analyzed by PCR–RFLP of 16S and 16S + IGS ribosomal regions, PCR–RFLP of six Ti plasmid (pTi) regions and characterized for plasmid content. Two groups of agrobacteria were separated by cluster analysis of PCR–RFLP profiles of rrs gene: seventeen strains were similar to the new species Agrobacterium larrymoorei, while the remaining strains were included within the agrobacterium biovar 1 group. Sixteen different plasmid profiles from one to five plasmids were observed. In addition, 21 ribotypes and 20 pTi structures were arranged in many different combinations, showing that fig agrobacteria were characterized by a wide heterogeneity. A general lack of correlation between strain ribotypes and plasmid content was observed.     

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。     

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.     

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.     

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.     

土壤杆菌K1026对樱桃冠瘿病病原菌的抑制作用及田间防治效果

由致病性根癌土壤杆菌Agrobacteriumspp.侵染引起的根癌病对樱桃等果树生产造成严重的影响。利用双层平板、胡萝卜圆盘法和接种指示植物番茄等方法验证了澳洲生防菌株K1026对2种不同原生物型樱桃冠瘿病病原菌的抑制作用;检测了不同接种时间对病原菌侵染产生瘤状组织的影响,结果显示提前或者在病原菌接种后20h内接种K1026可抑制胡萝卜产生瘤状组织;研究了K1026对大樱桃根癌病的作用机制是产生土壤杆菌素和竞争附着位点;田间试验表明K1026蘸根处理,对‘中国樱桃’株系冠瘿病的防治效果达81.61%,使发病率降低至18.13%。     

Horizontal gene transfer of atrazine-degrading genes (atz) from Agrobacterium tumefaciens St96-4 pADP1::Tn5 to bacteria of maize-cultivated soil

The plasmid pADP1::Tn5 derived from pADP1[Atr+] carrying a Tn5 transposon conferring kanamycin and streptomycin resistances was constructed and introduced in Agrobacterium tumefaciens St96-4. This genetically modified strain was inoculated (approximately 10(8) cfu g(-1)) in potted soils planted with maize and treated or not with atrazine (1.5 mg kg(-1)). Bulk and maize rhizosphere soils were sampled 39 days after planting to look for soil indigenous bacteria that had acquired pADP1::Tn5. Four transconjugants were isolated from four different soil samples. The estimated transfer frequency of pADP1::Tn5 was 10(-4) per donor. Maize rhizosphere and atrazine treatment had no obvious effect on pADP1::Tn5 transfer frequency. The sequencing of the 16S rDNA sequences of the transconjugants revealed that they were almost identical and highly similar to that of Variovorax spp (97%). In addition, their characterization suggested that the atzA and atzB genes had been transferred from pADP1::Tn5 to the bacterial chromosome in two of the four transconjugants. These data suggest that the atz degrading genes are horizontally transferred in soil and possibly subjected to gene rearrangement.     

Crown gall of tobacco caused by <Emphasis Type="Italic">Agrobacterium tumefaciens</Emphasis> biovar 1 in tobacco fields

Crown gall disease of tobacco was found in Iwate Prefecture, Japan in 1995. Ten bacterial isolates, obtained from the galls of tobacco, were identified as Agrobacterium tumefaciens (Smith and Townsend 1907) Conn 1942 biovar 1 based on their ability to induce galls on the 14 tested plants, including tobacco after needle-prick inoculation, and on 12 cultural, physiological, and biological characteristics. The growth of the causal organism was not inhibited in vitro by agrocin of A. radiobacter strain K84. This report is the first on the natural occurrence of crown gall caused by A. tumefaciens on tobacco plants.     

Physiological, Biochemical and Molecular Analyses of an Italian Collection of Agrobacterium tumefaciens strains

Physiological, biochemical and molecular characteristics of Agrobacterium tumefaciens strains isolated in Italy from different host plants were analysed. Diseased plants were collected from several nurseries located in nine different regions. Out of 1293 strains isolated from 12 fruit tree and six ornamental plant species, a group of 120 strains was chosen as representative of the whole collection. The majority of the strains were biovar 2 (82.5%), agrocin 84 sensitive, and were isolated from stone fruit trees. Most of the strains identified as biovar 1 were isolated from ornamental plants and were insensitive to A. radiobacter antagonistic strain K84. Some strains that were isolated from Euonymus spp, Prunus GF 677 and Pyrus communis (pear) OHF tumours could not be allocated to any of the three Agrobacterium biovars. PCR-restriction fragment length polymorphism of the rrs gene plus the intergenic spacer was used for strain fingerprinting and characterisation. Results showed a wide genetic variability within the biovar 1 strains and homogeneity within the biovar 2 group. Biovar 2 strains from Sardinia were highly variable and differed from the biovar 2 strains isolated from the other regions of Italy.     

Isolation and Characterization of a New Virulence Gene (abvA) of Agrobacterium tumefaciens

A mutant (M-1) was isolated by transposon (Tn5) insertion mutagenesis of Agrobacterium tumefaciens (strain A-208, C58 chromosome, nopaline type T37 pTi, virulent). The M-1 mutant exhibited a complete avirulent phenotype on Kalanchoe daigremontiana leaf and Kalanchoe pinnata stem but a very attenuated virulent phenotype on root of Daucus carota. The mutant had one insertion of Tn5 in pTi. A wild-type target segment (2.3 kb) that included the site of Tn5 insertion in M-1 mutant was cloned. Introducing the 2.3 kb segment into M-1 complemented completely the avirulent phenotype, producing galls as big as strain A-208. The 2.3 kb segment was sequenced, identifying three open reading frames, ORF 1 (354 bp), ORF 2 (261 bp) and ORF 3 (801 bp) in the segment. A Tn5 was inserted between the third and fourth nucleotide of ORF 1 in M-1. The ORF 1 had no homology to any reported genes and thus was named the abvA gene. The ORF 3 had the high homology (identities 44%, positive 68%) to the gene of the sarcosine oxidase β subunit (accession no. sp/P40875). Introduction of the DNA segment (743 bp) containing the abvA gene and its promoter region into M-1 partially complemented the avirulent phenotype of the mutant, producing galls smaller than strain A-208. The abvA gene was distributed not only on nopaline-type pTi (T37) but also on octopine-type pTi (A6NC) and chromosome (C58) of A. tumefaciens. M-1, being avirulent on K. daigremontiana and K. pinnata, had a Tn5 insertion only in the abvA gene on pTi but not in the abvA gene on the chromosome, implying that the abvA gene on the chromosome in strain A-208 is not functional. A binary vector, pIG121-Hm, containing the β -glucuronidase (GUS) gene with an intron was introduced into M-1, which was then applied to leaves of K. daigremontiana to assay GUS activity for monitoring T-DNA transfer to the host nucleus. High GUS activity comparable to that in strain A-208 was detected in M-1 in spite of its inability to induce galls, suggesting that M-1 can transfer T-DNA into the host nucleus, but cannot integrate it into the chromosome. Received 25 October 2000/ Accepted in revised form 28 December 2000     

Problems of Agrobacterium tumefaciens in planting material and its control1

Crown gall caused by Agrobacterium tumefaciens is a problem in many European nurseries especially on stone fruits, apples and some herbaceous hosts (biovars 1 and 2) and on grapevines (biovar 3). Nursery infection can arise from land infested with the pathogen. Disease incidence can be reduced to some extent by cultural methods such as crop rotation, minimizing wounding and selection of less susceptible cultivars. The best control of infection is the use of a pre-planting dip in the antagonist A. radiobacter strain 84, which gives excellent control of strains of the pathogen harbouring the nopaline-type Ti plasmid. The antagonist is not yet cleared for use in all European countries. This biological control is sometimes ineffective because of the presence of mixed populations of sensitive and resistant strains of the pathogen, use of too low inoculum doses of the antagonist or because the host plant affects the antagonist action. All controls are preventive and not curative, so latent infections in layer or stool beds cannot be prevented from developing. Control of the strains of the pathogen insensitive to strain 84 may be possible in future by the use of other antagonistic organisms or by genetic manipulation of strain 84. Until such a new development becomes available, growers must make careful selection of propagating material to minimize the risk of spreading infection.     

Rhizogenic Agrobacterium biovar 1 and cucumber root mat in the UK

An unusual root disease of cucumbers has affected an increasing number of hydroponic crops in the UK since 1993. Symptoms include upward growth of roots, root proliferation sufficient to distort the shape of rockwool cubes and slabs, thickened roots and occasionally reduced root production and an increase in bent fruit. Rhizogenic Agrobacterium biovar 1 has previously been associated with the problem. In a survey of 37 nurseries in 1997, rhizogenic Agrobacterium bv. 1 isolates were obtained from 17 of 22 nurseries with symptoms and only occasionally from nurseries without symptoms. Young cucumber plants inoculated with rhizogenic Agrobacterium bv. 1 isolates, previously obtained from cucumber roots with symptoms, developed typical symptoms. The survey data and pathogenicity test support rhizogenic Agrobacterium bv. 1 as the primary causal agent of root mat in the UK. Although Agrobacterium bv. 1 isolates that do not harbour an Ri-plasmid are considered avirulent, their abundance in rockwool slabs as potential vectors of the Ri-plasmid may be a major factor in the epidemiology of the disease.     

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。     

啤酒花根癌土壤杆菌生物型的鉴定及生物防治的初步探讨

啤酒花根癌病在我国发生普遍,是影响啤酒花生产的重要病害。从啤酒花主要生产基地新疆、山东、浙江等地分离到61个菌株。通过培养性状、染色反应,以及14～15个菌株的生理生化性状和12个菌株致病性的测定,确定啤酒花根癌病病原菌为根癌土壤杆菌Agrobacteria tumefaciens(E.F.Smith &Jownsend)Conn属生物型1、病原菌菌株对K84(Agrobacterium radiobacter)菌株的细菌素敏感,各菌株间敏感性的强弱不一。盆裁番茄和啤酒花初步生防试验表明,用K84与敏感性强的菌株按1:1比例混合接种,番茄和啤酒花都不产生瘿瘤;而敏感性弱的菌株需用3:1才能防止瘿瘤的产生。