In Vitro Binding of Agrobacterium tumefaciens to Plant Cells from Suspension Culture

In vitro binding experiments were carried out using (32)P-labeled cells of the virulent Agrobacterium tumefaciens strain B6 and Datura innoxia cells from suspension culture. Binding kinetics showed that adherence of bacteria to Datura cells increased gradually during the first 60 minutes and attained a maximum level within 120 minutes of incubation. Maximum binding occurred at pH 6.0. The presence of Ca(2+) and Mg(2+) reduced binding slightly and EDTA had little effect at concentrations of 0.1 to 10 millimolar. The binding of bacteria to Datura cells was temperature-dependent. Escherichia coli, Salmonella typhimurium, Rhizobium japonicum, and Micrococcus lysodeikticus did not compete with virulent A. tumefaciens strain B6 for binding to Datura cells. The admixture of avirulent A. tumefaciens strain IIBNV6 enhanced adherence of virulent A. tumefaciens strain B6 to Datura cells. Octopine had no effect on the binding of virulent A. tumefaciens strain B6 to Datura cells, but 10 millimolar canavanine was inhibitory. Arginine enhanced the adherence of the bacteria at concentrations higher than 0.1 millimolar. Incubation with DNase, RNase, and lipase did not affect the binding, but protease stimulated the adherence of bacteria to Datura cells. Concanavaline A and soybean lectin had little effect whereas lecithin and lysolecithin enhanced binding slightly. Poly-l-lysine markedly stimulated the bacteria-plant cell adherence. Cells from suspension cultures of pea, vetch, and soybean had a 2- to 3-fold higher binding capacity than Datura cells, whereas cells from wheat, corn, rice, and sorghum had a considerably lower affinity for binding with virulent A. tumefaciens strain B6. Bacterial adherence to plant cells was confirmed by autoradiography and electron microscopy. Autoradiographic analysis showed that bacteria were associated with the cell wall, and that often binding of bacteria was localized. Electron micrographs clearly illustrated a tight association of virulent A. tumefaciens strain B6 cells to the Datura cell wall.     

Biological Control of Agrobacterium tumefaciens, Colonization, and pAgK84 Transfer with Agrobacterium radiobacter K84 and the Tra Mutant Strain K1026

The efficacies of Agrobacterium radiobacter K84 and K1026 in root colonization, crown gall control, and plasmid transfer were compared. Levels of root colonization by K84 and K1026 of Montclar and Nemaguard peach seedlings were similar during the 21 days of the experiment. Four strains of A. tumefaciens bv. 1 were used for soil inoculations in biological control experiments on GF677 and Adafuel peach x almond rootstocks; two were sensitive and two were resistant to agrocin 84. Both strains K84 and K1026 were very efficient in controlling the sensitive strains, but some tumors appeared with both treatments. In the biocontrol of resistant strains, no galls were observed in K1026-treated plants, but some K84-treated plants had galls. Recovery of agrobacteria from galls in experiments with sensitive and resistant strains showed that all of the isolates from the controls or K1026-treated plants and most of the isolates from K84-treated plants had the same characteristics as the inoculated strains. Nine isolates from the K84-treated plants growing in soil inoculated with one resistant strain were virulent and produced agrocin 84. These isolates had a plasmid that hybridized with a probe prepared with the BamHI C fragment from pAgK84. These results show the efficiency of K1026 in biocontrol of agrocin 84-sensitive and -resistant strains of A. tumefaciens and suggest the use of K1026 as a safer organism than K84 for biological control of crown gall.     

日本樱花根癌病病原菌的鉴定及其防治

从浙江省的慈溪、奉化、嵊州等地的日本樱花苗圃内,采集到具有典型症状的日本樱花根癌病植株。经分离纯化及在指示植物番茄、向日葵幼苗的致病性测定,共分离到致病性病原菌株１１株,经形态学、生理生化学特征鉴定及菌体可溶性蛋白ＳＤＳ－聚丙烯酰胺凝胶电泳,确定引起日本樱花根癌病的病原细菌为根癌土壤杆菌（Ａｇｒｏｂａｃｔｅｒｉｕｍｔｕｍｅｆａｃｉｅｎｓ）生物型１,经平皿拮抗和盆栽试验表明,生防菌Ｋ８４能够明显抑制致病菌株的致癌能力。     

Biological control of crown gall in cherry rootstock propagation

English isolates of Agrobacterium tumefaciens from cherry were sensitive to the bacteriocin produced by A. radiobacter strain 84 in vitro , and simultaneous inoculation of the two organisms into tomato stems or cherry leaf scars completely inhibited the gall formation that occurred in the absence of strain 84. However, attempts to achieve biological control of crown gall of cherry in the field were successful only when the antagonist was applied as a preplanting treatment to cuttings. Disease on infected cherry layers was not reduced even after three sprays of the antagonist. A. radiobacter strain 84 was also ineffective as a preplanting dip, or both preplanting and post-harvest dips for symptomless, but latently infected, rootstocks harvested from an infected layer bed.     

Conjugation in Agrobacterium tumefaciens in the absence of plant tissue.

A general, reliable conjugation system for Agrobacterium tumefaciens in the absence of plant tissue is described in which A. tumefaciens can serve either as the donor or recipient of plasmid deoxyribonucleic acid with reasonable efficiency. Plasmid RP4 was transferred from Escherichia coli to A. tumefaciens and from strain of A. tumefaciens. Both RP4 and the A. tumefaciens virulence-associated plasmids were detected by alkaline sucrose gradients in A. tumefaciens strains A6 and C58 after mating with E. coli J53(RP4). The pathogenicity (tumor foramtion) of strains A6 and C58 and the sensitivity of strain C58 to bacteriocin 84 were unaffected by the acquistion of RP4 by the Agrobacterium strains. Plasmid R1drd-19 was not transferred to A. tumefaciens. Transformation experiments with plasmid deoxyribonucleic acid were unsuccessful, even though, in the case of RP4, conjugation studies showed taht the deoxyribonucleic acid was compatible with that of the recipient strains.     

Transfer of nitrogen fixation genes from a bacterium with the characteristics of both Rhizobium and Agrobacterium.

Strain T1K, reported to be Rhizobium trifolii strain T1 carrying the drug resistance plasmid RU-1drd, was able to transfer a cluster of nif+ genes to Escherichia coli K-12. Additional genetic material, resembling the gal-chlA region of E. coli, was also transferred from strain T1K. The segregation pattern of these transferred genes suggested that they were on a plasmid. Although strain TIK was able to nodulate red and white clover, it also formed very slow-growing galls on tomato stems and shared many physiological properties with Agrobacterium tumefaciens, to which it seemed more closely related than to R. trifolii. The R. trifolii hybrid T1 (R1-19drd), constructed by conjugation, did not share any of these properties of both A. tumefaciens. Thus, strain T1K appears to be a bacterium with properties of both A. tumefaciens and R. trifolii and with the capacity to transfer nif+ genes and other functions which it may have "cloned" from another bacterium such as Klebsiella.     

Host-Bacteriophage Interaction in Agrobacterium tumefaciens III. Phage-Coded Endolysins

Endolysins were detected in a sensitive strain of Agrobacterium tumefaciens (B6) after infection with phage LV-1 and in the lysogen A. tumefaciens V-1 after induction with mitomycin C. A similar endolysin was found in mitomycin C-induced A. tumefaciens C-58, which apparently harbors a defective prophage.     

Transformation of Cucumis melo cv. Hetau with Tomato Antisense ACC Synthase Gene

An efficient in vitro plant regeneration system of Cucumis melo L. cv. Hetau was established. Regenerated plantlets were obtained from cotyledons after preculture, shoot inducing culture and root inducing culture. A high regeneration rate was achieved up to 58%. Cucumis melo was transformed with the antisense tomato ACC synthase gene in binary vector pMQ6 via Agrobacterium tumefaciens mediated gene transfer. Kanamycin resistant plantlets were obtained on MS medium with 6 mg/L zeatin, 50 mg/L kanamycin and 650 mg/L cefotaximine. PCR and molecular hybridization analysis showed that tomato ACC synthase antisense cDNA was integreted into the genome of C. melo.     

Deletion derivatives of pAgK84 and their use in the analysis of Agrobacterium plasmid functions.

The 47.7-kb plasmid pAgK84, present in Agrobacterium radiobacter strain K84, confers production of a novel, highly specific, antiagrobacterial antibiotic called agrocin 84. Strain K84 is used commercially to biocontrol crown gall caused by agrocin 84-susceptible strains of Agrobacterium tumefaciens. Efficient biocontrol is dependent upon production of agrocin 84 by strain K84. Starting with a derivative of pAgK84 containing a Tn5 insertion, a series of deletion derivatives of the plasmid were isolated. The smallest of these, pJS500, contains about 8 kb of the original agrocin plasmid and localized the replication functions to between 4 and 6 o'clock on the physical map. A smaller derivative, produced by clonal rescue of a Tn5 insertion in the 4 o'clock region, further localized the minimal replication functions to a 1.5-kb region mapping between coordinates 18.1 and 19.6. Analysis of plasmid stability indicated that functions required for maintenance of the plasmid under nonselective conditions are tightly linked to the minimal replication region. This region also encodes incompatibility functions; the deletion derivatives were all incompatible with the wild-type pAgK84. The stability/replication locus of pAgK84 maps just anticlockwise from the Tra region. This region is retained fully in pAgK1026, the directed Tra- derivative of pAgK84 which is now in use as the primary crown gall biocontrol agent in Australia. One of the deletion derivatives, the 15-kb pJS400, was used as a vector to clone the KpnI fragments of an octopine-type Ti plasmid. Traits known to be encoded on these fragments were expressed and properly regulated in Agrobacterium hosts. One clone, encoding the Ti plasmid replication/incompatibility region, was used to cure IncRh1 Ti plasmids from their hosts. This clone also was found to be incompatible with pAtK84b, a large plasmid encoding opine catabolism present in A. radiobacter strain K84. This indicates that the opine catabolic plasmid is closely related to the IncRh1 Ti plasmids.     

The effect of cellulose overproduction on binding and biofilm formation on roots by Agrobacterium tumefaciens

Agrobacterium tumefaciens growing in liquid attaches to the surface of tomato and Arabidopsis thaliana roots, forming a biofilm. The bacteria also colonize roots grown in sterile quartz sand. Attachment, root colonization, and biofilm formation all were markedly reduced in celA and chvB mutants, deficient in production of cellulose and cyclic beta-(1,2)-D-glucans, respectively. We have identified two genes (celG and cell) in which mutations result in the overproduction of cellulose as judged by chemical fractionation and methylation analysis. Wild-type and chvB mutant strains carrying a cDNA clone of a cellulose synthase gene from the marine urochordate Ciona savignyi also overproduced cellulose. The overproduction in a wild-type strain resulted in increased biofilm formation on roots, as evaluated by light microscopy, and levels of root colonization intermediate between those of cellulose-minus mutants and the wild type. Overproduction of cellulose by a nonattaching chvB mutant restored biofilm formation and bacterial attachment in microscopic and viable cell count assays and partially restored root colonization. Although attachment to plant surfaces was restored, overproduction of cellulose did not restore virulence in the chvB mutant strain, suggesting that simple bacterial binding to plant surfaces is not sufficient for pathogenesis.     

Agrocin 84 sensitivity: a plasmid determined property in Agrobacterium tumefaciens.

It was shown for some oncogenic Agrobacterium tumefaciens strains that agrocin 84 sensitivity is determined by the presence of a large closed circular DNA plasmid, called the Ti-plasmid. Whereas wild-type strain C58 is agrocin 84 sensitive, all Ti-plasmid cured derivatives were found to be fully resistant. Moreover all independently isolated agrocin 84 resistant colonies were stably non-oncogenic and plasmid negative. In a growth experiment carried out at 37 degrees C it was shown that the kinetics of appearance of non-oncogenic cells on the one hand and of agrocin 84 resistant cells on the other were identical. The fact that not all oncogenic, plasmid harbouring, Agrobacterium tumefaciens strains are sensitive to agrocin 84, points to the possibility that the genes determining agrocin 84 sensitivity are not essential for tumor-inducing ability.     

Plasmid required for virulence of Agrobacterium tumefaciens.

The irreversible loss of crown gall-inducing ability of Agrobacterium tumefaciens strain C-58 during growth at 37 C is shown to be due to loss of a large plasmid (1.2 X 10-8 daltons). The gene responsible for this high rate of plasmid loss at elevated temperatures seems to be located on the plasmid. In addition, another spontaneous avirulent variant, A. tumefaciens strain IIBNV6 is shown to lack the virulence plasmid which its virulent sibling strain, IIBV7, possesses. Deoxyribonucleic acid reassociation measurements prove that the plasmid is eliminated, not integrated into the chromosome, in both of the avirulent derivatives. Transfer of virulence from donor strain C-58 to avirulent recipient strain A136 results from the transfer of a plasmid, which appears identical to the donor plasmid by deoxyribonucleic acid reassociation measurements. The transfer of virulence in another cross, K27 X A136, was also shown to result from the transfer of a large plasmid. These findings establish unequivocally that the large plasmid determines virulence. Two additional genetic determinants have been located on the virulence plasmid of A. tumefaciens strain C-58: the ability to utilize nopaline and sensitivity to a bacteriocin produced by strain 84. The latter trait can be exploited for selection of avirulent plasmid-free derivatives of strain C-58. The trait of nopaline utilization appears to be on the virulence plasmid also in strains IIBV7 and K27.     

由根癌农杆菌介导向毛白杨导入激素合成基因建立遗传转化系统

用携带基因1,2的根癌农杆菌AG(84)转化毛白杨外植体,在无激素的MS0培养基上获得转化根。分离单根或切成根段在分化培养基上能分化芽而再生完整植株。由T－DNA上带有基因4的根癌农杆菌C58C1（PBZ6111）转化毛白杨外植体,在MS0培养基上能直接分化不定芽而再生植株．在转化中使用叶柄作外植体比使用叶片的转化率提高一倍以上。基因1,2引入毛白杨后,植株根系发达,生根率达100％。基因4引入毛白杨则使植株节间变短,植株矮化．纸电泳分析表明,带有基因1,2的转化植株能表达特异的农杆碱,带有基因4的转化植株能表达特异的胭脂碱。     

Aeromonas, a possible etiological agent in the formation of secondary tumors of crown gall

Abstract From a secondary tumor in a bean stem we have isolated a Gram-negative bacteria, named by us T.2. These bean stems had crown gall tumors induced by the ATV strain of Agrobacterium tumefaciens . This bacterium was classified as belonging to the genus Aeromonas and possesses the capacity of inducing overgrowths in plants, synthesizing indole acetic acid (IAA). The codified phenotypic characteristics of bacterium T.2. via the Ti-plasmid of A. tumefaciens , such as opine utilization and sensitivity to agrocin 84, have been studied. Neither octopine nor nopaline is utilized by T.2. and it is resistant to agrocin 84, whereas the strain ATV of A. tumefaciens utilizes nopaline, and is sensitive to agrocin 84.     

Monoxenic maintenance and reproduction of root-knot nematode (Meloidogyne hapla) on multiple-species in vitro root culture systems

The ability of tomato root tips transformed with Agrobacterium rhizogenes and non-transformed onion and dandelion root tips to regenerate and provide suitable monoxenic hosts for the maintenance of root-knot nematode (Meloidogyne hapla) was investigated. Populations of M. hapla were successfully established on all root culture systems examined, but the onion root culture was overall the most effective method for long-term maintenance and reproduction of M. hapla. The use of a pre-induction medium containing the hormone !-naphthaleneacetic acid was necessary for the production of onion root culture systems but was not required for the establishment of tomato or dandelion root cultures. Differences in nematode reproduction were observed on tomato when multiple populations of M. hapla were used for inoculations.     

The Agrobacterium tumefaciens virD3 gene is not essential for tumorigenicity on plants.

Genetic studies indicate that three of the four polypeptides encoded within the virD operon of the Agrobacterium tumefaciens Ti plasmid are essential for virulence. In order to determine whether the fourth polypeptide, VirD3, has any role in virulence, complementation analysis was used. An A. tumefaciens strain, A348 delta D, which lacked the entire virD operon in the Ti plasmid pTiA6, was constructed. Plasmids containing defined regions of the virD operon were introduced into this strain, and virulence was tested by the strains' abilities to form tumors on Kalanchoe leaves, tomato stems, and potato tubers. As expected, deletion of the virD operon led to an avirulent phenotype. The virulence of this strain could be restored by providing virD1, virD2, and virD4 in trans. No requirement for virD3 in tumor formation was observed in these assays.     

Fate of Agrobacterium radiobacter K84 in the environment.

Agrobacterium radiobacter K84 is an effective, commercially applied, biological control agent for the plant disease crown gall, yet little is known about the survival and dissemination of K84. To trace K84 in the environment, spontaneous antibiotic-resistant mutants were used. Growth rates and phenotypes of streptomycin- or rifampin-resistant K84 were similar to those of the parental K84, except the rifampin-resistant mutant produced less agrocin 84 as determined by bioassay. K84 and a strain of Agrobacterium tumefaciens established populations averaging 10(5) CFU/g in the rhizosphere of cherry and persisted on roots for 2 years. K84 established rhizosphere populations between 10(4) and 10(6) CFU/g on cherry, ryegrass, and 11 other herbaceous plants. Populations of K84 declined substantially in fallow soil or water over a 16-week period. K84 was detected in the rhizosphere of ryegrass located up to 40 cm from an inoculum source, indicating lateral dissemination of K84 in soil. In gall tissue on cherry, K84 established populations of 10(5) CFU/g, about 10- to 100-fold less than that of the pathogen. These data demonstrate that K84 persists for up to 2 years in a field environment as a rhizosphere inhabitant or in association with crown gall tissue.     

Agrobacterium radiobacter strains K84, K1026 and K84 Agr‐ produce an antibiotic‐like substance,active in vitro against A. tumefaciens and phytopathogenic Erwinia and Pseudomonas spp.

Agrobacterium radiobacter strains K84, K1026 and K84 Agr^‐ produced in vitro an antibiotic‐like substance (ALS 84), different from agrocin 84 and observed in mannitol‐glutamate medium. Twenty five out of 39 A. tumefaciens strains of biovars 1, 2 and 3 were sensitive to ALS 84 regardless of their sensitivity to agrocin 84. Sensitivity in A. tumefaciens strain C58 was not encoded by the Ti‐plasmid. Most isolates tested of Erwinia carotovora subsp. carotovora E. carotovora subsp. atroseptica, Pseudomonas corrugata P. cichorii and unidentified isolates from galls were also sensitive to this substance. ALS 84 was not affected by the proteases studied, nor by treatment at 62°C for 30 min and had a bacteriostatic effect. The production of ALS 84 might play a role in the complex mechanism of biological control of crown gall, especially in strains resistant to agrocin 84 and sensitive to ALS 84, and by the creation of an ecological niche favourable to A. radiobacter strains K84, K1026 or K84 Agr^‐.     

Mobilization and transfer of Azospirillum lipoferum plasmid by the Tn5-Mob transposon into a plasmid-free Agrobacterium tumefaciens strain

Azospirillum lipoferum 4B harbors five cryptic plasmids. Several suicide plasmids were used to transfer Tn5-Mob to A. lipoferum 4B. Tn5-Mob insertion mutations of this strain could be obtained at frequencies of 10(-8)-10(-7) per recipient cell. One hundred Tn5-Mob A. lipoferum 4B mutants were used in bacterial matings with a plasmid-free Agrobacterium tumefaciens recipient strain. This is the first report of mobilization, transfer, and replication of an Azospirillum plasmid in Agrobacterium tumefaciens. One transconjugant was found which had lost an indigenous plasmid.     

Interaction between Meloidogyne incognita and Agrobacterium tumefaciens or Fusarium oxysporum f. sp. lycopersici on Tomato

Agrobacterium tumefaciens stimulated and Fusarium oxysporum f. sp. lycopersici inhibited development and reproduction of Meloidogyne incognita when applied to the opposite split root of tomato, Lycopersicon esculentum cv. Tropic, plants. The lowest rate of nematode reproduction occurred after 2,000 juveniles were applied and the fungus was present in the opposite split root. The effects of all three pathogens alone on the growth of roots and shoots of tomato plants were evident, but M. incognita had a greater effect alone than did either of the other pathogens. The length of split roots was reduced by the infection of M. incognita and A. tumefaciens or F. oxysporum f. sp. lycopersici. The number of galls induced by nematodes on roots was higher where the bacterium was applied and lower where the fungus was applied to the opposite split root.