Evidence of Biological Control of Agrobacterium tumefaciens Strains Sensitive and Resistant to Agrocin 84 by Different Agrobacterium radiobacter Strains on Stone Fruit Trees

The effectiveness of Agrobacterium radiobacter K84, 0341, and a K84 non-agrocin-producing mutant (K84 Agr^-) in biological control of crown gall on rootstocks of stone fruit trees was determined in three experiments. In experiment 1, K84 and 0341 controlled crown gall on plum plants in soil inoculated with two strains of Agrobacterium tumefaciens resistant to agrocin 84. In experiment 2, K84 controlled crown gall on peach plants in soils inoculated with strains of A. tumefaciens sensitive or resistant to agrocin 84 or with a mixture of both. However, the effectiveness of K84 was higher against the sensitive strain than against the resistant strain. There was a residual effect of K84 from one year to another in soil inoculated with the sensitive strains. In experiment 3, K84 and K84 Agr^- controlled crown gall on plum and peach plants in soils inoculated with strains of A. tumefaciens sensitive or resistant to agrocin 84. The control afforded by K84 was higher than that provided by K84 Agr^- against the sensitive strain but was similar against the resistant strain.     

Broad-Host-Range Agrocin of Agrobacterium tumefaciens

Eighteen strains of Agrobacterium tumefaciens isolated from crown galls were tested for agrocin production. Of six agrocin-producing strains, one (D286) produced a broad-host-range agrocin active against strains carrying nopaline, octopine, and agropine type Ti plasmids. Sensitivity to agrocin D286 was found to map in the 11- to 18-megadalton region of the nopaline Ti plasmid pTiC58. The agrocin was partially purified, and its physical characteristics were consistent with its being a nucleotide, as is agrocin 84. Agrocin D286 was shown to inhibit DNA, RNA, and protein syntheses. Strain D286 spontaneously lost its pathogenicity, and its potential for use in the biological control of crown gall is discussed.     

Cocolonization of the Rhizosphere by Pathogenic Agrobacterium Strains and Nonpathogenic Strains K84 and K1026, Used for Crown Gall Biocontrol

The crown gall biocontrol agent strain K84 and three mutants derived from it, K1026 (Tra⁻ deletion mutant of pAgK84), K84 Agr⁻ (lacking pAgK84), and K1143 (lacking pAgK84 and pNoc), significantly reduced gall formation caused by two pathogenic strains resistant to agrocin 84 in peach × almond seedlings planted in infested soil. Cocolonization of roots by pathogenic and nonpathogenic strains was observed in these biocontrol experiments under field conditions. In spite of the efficient biocontrol observed, average populations consisting of 10² and 10⁶ pathogenic agrobacteria per g of root were found 8 months after planting. The total numbers of pathogenic bacteria on roots were similar for plants treated with the biocontrol strains and for the untreated plants. Strain K84 and the genetically engineered organism K1026 survived at a level of 10⁶ agrocin 84-producing bacteria per g of root. The population size of genetically engineered strain K1026 was not significantly different than the population size of wild-type strain K84 8 months after root inoculation. Strains K84 and K1026 controlled two pathogens resistant to agrocin 84 without reducing the total number of pathogenic bacteria in the root system. In addition, this study shows that some biological control activity of strain K84 against agrocin 84-resistant pathogens is independent of plasmids pAgK84 and pNoc.     

Susceptibility of Agrobacterium tumefaciens Strains to Two Agrocin-Producing Agrobacterium Strains

Sixty-five strains and isolates of Agrobacterium tumefaciens representing each of the known biotypes, were tested for in vitro and in vivo susceptibility to the agrocin-producing strains Agrobacterium radiobacter 84 and A. tumefaciens D286. No biotype 3 strain was susceptible to the effects of either of the agrocinogenic strains in vitro. On datura and tobacco, the best inhibition of tumor formation was obtained when the agrocinogenic strains were applied to wounds 24 h before the pathogens and by the concomitant application of agrocin producer and pathogen at cell ratios of 10:1 or 3:1; inhibition of infection tended to decrease progressively as the cell ratio decreased from 10:1 to 3:1 to 1:1. Generally, strain 84 was superior to D286 in inhibiting tumor formation. A combined cell suspension of 84 and D286 was as effective as 84 alone. The overall pattern of inhibition of tumor formation by biotype 1 and 2 pathogens resistant to the agrocinogenic strains in vitro was similar to that obtained with strains that were susceptible in vitro.     

Genetic analysis of an Agrobacterium tumefaciens strain producing an agrocin active against biotype 3 pathogens

Summary The successful biocontrol agent for crown gall disease, Agrobacterium radiobacter strain K84, is unable to protect grapevines from infection. We have identified a strain of Agrobacterium tumefaciens, J73, which produces an agrocin active both in vitro and in vivo against grapevine pathogens (Webster et al. 1986). We now report on the curing of this strain of its nopaline-type Ti plasmid and the location, by transposon mutagenesis, of the genes involved in the production of the agrocin. The Ti plasmid was cured by the introduction of selectable plasmids carrying the origins of replication of either the nopaline Ti plasmid, pTiC58, or the octopine Ti plasmid, pTi15955. Tn5 mutagenesis indicated that the genes responsible for agrocin production and/or export are located both on the chromosome and on a plasmid, pAgJ73, which co-migrated in agarose gels with pTiJ73. As the two plasmids were separable after transposon mutagenesis, we postulate that during or after mutagenesis of the agrocin plasmid, DNA rearrangements occurred between it and pTiJ73, resulting in an increase in size of pAgJ73. We provide evidence that the rearrangements involved the duplication of nopaline catabolism genes from pTiJ73 and their insertion into pAgJ73, which facilitated the resolution of the two plasmids. As expected pTiJ73 has homology with the nopaline Ti plasmid, pTiC58.     

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^‐.     

Location and Survival of Leaf-Associated Bacteria in Relation to Pathogenicity and Potential for Growth within the Leaf

The growth and survival of pathogenic and nonpathogenic Pseudomonas syringae strains and of the nonpathogenic species Pantoea agglomerans, Stenotrophomonas maltophilia, and Methylobacterium organophilum were compared in the phyllosphere of bean. In general, the plant pathogens survived better than the nonpathogens on leaves under environmental stress. The sizes of the total leaf-associated populations of the pathogenic P. syringae strains were greater than the sizes of the total leaf-associated populations of the nonpathogens under dry conditions but not under moist conditions. In these studies the surface sterilants hydrogen peroxide and UV irradiation were used to differentiate cells that were fully exposed on the surface from nonexposed cells that were in “protected sites” that were inaccessible to these agents. In general, the population sizes in protected sites increased with time after inoculation of plants. The proportion of bacteria on leaves that were in protected sites was generally greater for pathogens than for nonpathogens and was greater under dry conditions than under moist conditions. When organisms were vacuum infiltrated into leaves, the sizes of the nonexposed “internal” populations were greater for pathogenic P. syringae strains than for nonpathogenic P. syringae strains. The sizes of the populations of the nonpathogenic species failed to increase or even decreased. The sizes of nonexposed populations following spray inoculation were correlated with the sizes of nonexposed, internal populations which developed after vacuum infiltration and incubation. While the sizes of the populations of the pathogenic P. syringae strains increased on leaves under dry conditions, the sizes of the populations of the nonpathogenic strains of P. syringae, P. agglomerans, and S. maltophilia decreased when the organisms were applied to plants. The sizes of the populations on dry leaves were also correlated with the sizes of the nonexposed populations that developed following vacuum infiltration. Although pathogenicity was not required for growth in the phyllosphere under high-relative-humidity conditions, pathogenicity apparently was involved in the ability to access and/or multiply in certain protected sites in the phyllosphere and in growth on dry leaves.     

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.     

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 × 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.     

Colonization of Tomato Plants by Two Agrocin-Producing Strains of Agrobacterium tumefaciens

For a bacterium to be a successful biocontrol agent against crown gall disease, it must produce an effective agrocin specific for Agrobacterium tumefaciens and be able to colonize host plants efficiently. The colonization abilities of K84 and J73, successful and potential biocontrolling strains, respectively, were compared both in vivo and in vitro. Both strains produced fibrils attaching them to tomato root surfaces and had similar colonization efficiencies up to 14 days after inoculation. However, the ability of J73 to colonize plants for longer periods was significantly less than that of K84. Thus, the presence of fibrils is not sufficient to ensure colonization. No correlation was found between hydrophobicity and colonization.     

Influence of Plant Root Exudates, Germ Tube Orientation and Passive Conidia Transport on Biological Control of Fusarium Wilt by Strains of Nonpathogenic Fusarium oxysporum

In earlier studies, biological control of Fusarium wilt of cucumber induced by Fusarium oxysporum f. sp. cucumerinum was demonstrated using nonpathogenic strains C5 and C14 of Fusarium oxysporum. Strain C14 induced resistance and competed for infection sites whether roots were wounded or intact, whereas strain C5 required wounds to achieve biocontrol. In the current work, additional attributes involved in enhanced resistance by nonpathogenic biocontrol agents strains to Fusarium wilt of cucumber and pea were further investigated. In pre-penetration assays, pathogenic formae specials exhibited a significantly higher percentage of spore germination in 4-day-old root exudates of cucumber and pea than nonpathogens. Also, strain C5 exhibited the lowest significant reduction in spore germination in contrast to strain C14 or control. One-day-old cucumber roots injected with strain C14 resulted in significant reduction in germ tube orientation towards the root surface, 48–96 h after inoculation with F. o. cucumerinum spores, whereas strain C5 induced significantly lower spore orientation of the pathogen and only at 72 and 96 h after inoculation. In post-penetration tests, passive transport of microconidia of pathogenic and nonpathogens in stems from base to apex were examined when severed plant roots were immersed in spore suspension. In repeated trials, strain C5, F. o. cucumerinum and F. o. pisi were consistently isolated from stem tissues of both cucumber and pea at increasing heights over a 17 days incubation period. Strain C14 however, was recovered at a maximum translocation distance of 4.6 cm at day 6 and later height of isolation significantly declined thereafter to 1.2 cm at day 17. In pea stem, the decline was even less. Significant induction of resistance to challenge inoculation by the pathogen in cucumber occurred 72 and 96 h after pre-inoculation with biocontrol agents. Nonetheless, strain C14 induced protection as early as 48 h and the maximum resistance was reached at 96 h. The presented data confirm the previous findings that attributes important for nonpathogenic fusaria to induce resistant are: rapid spore germination and orientation in response to root exudate; active root penetration and passive conidia transport in stem to initiate defence reaction without pathogenicity and enough lag period between induction and challenge inoculation. Strain C14 possesses all these qualifications and hence its ability to enhance host resistance is superior than strain C5.     

Evaluation of endophytic actinobacteria as antagonists of Gaeumannomyces graminis var. tritici in wheat

Endophytic actinobacteria isolated from healthy cereal plants were assessed for their ability to control fungal root pathogens of cereal crops both in vitro and in planta. Thirty eight strains belonging to the genera Streptomyces, Microbispora, Micromonospora, and Nocardioidies were assayed for their ability to produce antifungal compounds in vitro against Gaeumannomyces graminis var. tritici (Ggt), the causal agent of take-all disease in wheat, Rhizoctonia solani and Pythium spp. Spores of these strains were applied as coatings to wheat seed, with five replicates (25 plants), and assayed for the control of take-all disease in planta in steamed soil. The biocontrol activity of the 17 most active actinobacterial strains was tested further in a field soil naturally infested with take-all and Rhizoctonia. Sixty-four percent of this group of microorganisms exhibited antifungal activity in vitro, which is not unexpected as actinobacteria are recognized as prolific producers of bioactive secondary metabolites. Seventeen of the actinobacteria displayed statistically significant activity in planta against Ggt in the steamed soil bioassay. The active endophytes included a number of Streptomyces, as well as Microbispora and Nocardioides spp. and were also able to control the development of disease symptoms in treated plants exposed to Ggt and Rhizoctonia in the field soil. The results of this study indicate that endophytic actinobacteria may provide an advantage as biological control agents for use in the field, where others have failed, due to their ability to colonize the internal tissues of the host plant.     

Occurrence of free and conjugated 12,13-epoxytrichothecenes and zearalenone in banana fruits infected with Fusarium moniliforme.

Agrobacterium tumefaciens J73, a biotype 2 strain harboring a nopaline Ti plasmid, was found to produce an agrocin active against a broad range of A. tumefaciens strains, including grapevine isolates. Sensitivity to J73 is not encoded by a Ti plasmid. Optimal conditions for the production of the agrocin were determined.     

Bile-mediated aminoglycoside sensitivity in Lactobacillus species likely results from increased membrane permeability attributable to cholic acid

Few studies have been conducted on antimicrobial resistance in lactobacilli, presumably because of their nonpathogenic nature as anaerobic commensals. We assessed resistance in 43 type strains and isolates representing 14 species by using agar disk diffusion and MIC analysis in MRS medium. Most noteworthy were two general phenotypes displayed by nearly every strain tested: (i) they were more susceptible (up to 256-fold in some cases) to the deconjugated bile acid cholic acid than to the conjugate taurocholic or taurodeoxycholic acid, and (ii) they became susceptible to aminoglycosides when assayed on agar medium containing 0.5% fractionated bovine bile (ox gall). Two-dimensional MIC analyses of one representative strain, Lactobacillus plantarum WCFS1, at increasing concentrations of ox gall (0 to 30.3 mg/ml) displayed corresponding decreases in resistance to all of the aminoglycosides tested and ethidium bromide. This effect was clinically relevant, with the gentamicin MIC decreasing from >1,000 to 4 mug/ml in just 3.8 mg of ox gall per ml. In uptake studies at pH 6.5, [G-3H]gentamicin accumulation increased over control levels when cells of this strain were exposed to bile acids or reserpine but not when they were exposed to carbonyl cyanide m-chlorophenylhydrazone. The effect was dramatic, particularly with cholic acid, increasing up to 18-fold, whereas only modest increases, 3- and 5-fold, could be achieved with taurocholic acid and ox gall, respectively. Since L. plantarum, particularly strain WCFS1, is known to encode bile salt hydrolase (deconjugation) activity, our data indicate that mainly cholic acid, but not taurocholic acid, effectively permeabilizes the membrane to aminoglycosides. However, at pHs approaching neutral conditions in the intestinal lumen, aminoglycoside resistance due to membrane impermeability may be complemented by a potential efflux mechanism.     

Acanthamoeba Can Be Differentiated by the Polymerase Chain Reaction and Simple Plating Assays

Acanthamoeba are opportunistic pathogens with invasive and noninvasive species. For clinical purposes it is important to differentiate potentially pathogenic from nonpathogenic isolates. For the rapid and sensitive identification of Acanthamoeba at the genus level, we used a polymerase chain reaction (PCR)-based method which detected as few as five cells. Further, we tested nine isolates of Acanthamoeba for their ability to produce cytopathic effects (CPE) on corneal epithelial cells. On the basis of the results, Acanthamoeba were divided into pathogenic or nonpathogenic groups. However, because CPE assays are not available to every diagnostic laboratory, we developed a simple plating assay based on osmotolerance which correlated well with the CPE assays. Pathogenic Acanthamoeba showed growth on higher osmolarity (agar plates containing one molar mannitol), while growth of nonpathogens was inhibited on these plates. In conclusion, we have developed methods for the rapid identification and differentiation of Acanthamoeba. Received: 5 January 2001/Accepted: 6 February 2001     

Induction of lytic enzymes by the interaction of Ustilago maydis with Zea mays tissues

The nonpathogenic (FB-2) and pathogenic (FB-D12) strains of Ustilago maydis were grown in medium supplemented with different carbon sources including monosaccharides, polysaccharides, and plant tissues. Both strains were able to grow on all substrates, with doubling times varying from 2 to 25 h depending on the carbon source. Plant tissues supplied as carbon source induced lytic enzymes differentially; pectate lyase and cellulase activities were induced preferentially by apical stem meristem in strain FB-D12, whereas leaves preferentially induced xylanase and cellulase activities in strain FB2. Stems induced polygalacturonase activity in both strains. All enzyme activities, except cellulase in the FB-D12 strain, were detected at a low level when U. maydis was grown on glucose. In planta, chlorosis and production of teliospores were paralleled by an increase in pectate lyase activity. Anthocyanin production and formation of galls and teliospores correlated with polygalacturonase expression whereas cellulase activity increased only during the stage of anthocyanin production and gall formation. Expression of xylanase activity coincided with the last stage of teliospore formation.     

New Antagonistic Strains of Non‐Pathogenic Agrobacterium vitis to Control Grapevine Crown Gall

Graft unions of nursery stock of grapevine (Vitis vinifera L.) collected in Japan yielded non‐pathogenic strains of Agrobacterium. On the basis of classic diagnostic tests, a sequence analysis and a previously reported multiplex PCR method, the non‐pathogenic strains ARK‐1, ARK‐2 and ARK‐3 were identified as Agrobacterium vitis. Stems of grapevine seedlings were inoculated with both a cell suspension of seven mixed strains of A. vitis (Ti) as a pathogen and one of a new strain or A. vitis strain VAR03‐1, one of the biological control agents against crown gall previously reported, as competitors to assay the suppression of tumour formation caused by the pathogen. In a test with a 1:1 cell ratio of pathogen/nonpathogen, strains ARK‐1, ARK‐2 and ARK‐3 reduced the tumour incidence.. In particular, strain ARK‐1 was strongest at inhibiting tumour formation in this study. Strain ARK‐1 established populations on roots of grapevine tree rootstock and persisted on roots for a year. ARK‐1, ARK‐2 and ARK‐3 did not produce a halo of inhibition against A. vitis (Ti) strain on YMA medium. Moreover, strain ARK‐1 did not reduce tumour incidence on the stems of grapevine when ARK‐1 was dead or only culture filtrate was used. This result indicates the possibility that these new strains inhibit grapevine crown gall in planta by a different mechanism other than VAR03‐1. In particular, one of the new strains, named ARK‐1, was most effective in inhibiting tumour formation on grapevine and appears to be a promising new agent to control grapevine crown gall.     

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.