Pseudomonas Fluorescens Strain A506 Research Articles

Effects of Bacteria and Compost Tea Applications on Fruit Characteristics, Yield and Nutrient Content on Salihli Sweet Cherry Variety

Amaç: Bu çalışmada, Salihli kiraz çeşidinde bakteri ve kompost çayı uygulamalarının meyve özellikleri, verim, gelişme ve besin elementi içeriklerine etkisinin araştırılması amaçlanmıştır. Materyal ve Metot: Araştırmada P. mahaleb anacına aşılı Salihli kiraz çeşidi bitkisel materyal olarak kullanılmıştır. Pantoae agglomerans strain C9/1 ve Pseudomonas fluorescens strain A506 bakterileri ağaçlara 3 farklı dönemde (tam çiçeklenme, çiçeklenmeden 15 ve 30 gün sonar) kompost çayı ise 15 günlük periyotlarda (yapraklanma - meyvelerde renk dönüşümü) uygulanmıştır. Sürgün uzunluğu, meyve özellikleri, makro ve mikro besin elementleri ve verim incelenmiştir. Bulgular: A506 bakteri uygulaması ile sürgün uzunluğu ve sap kopma kuvveti artış göstermiştir. Bakteri ve kompost çayı uygulamalarında, meyvelerin koyu kırmızı renkli olduğu belirlenmiştir. Uygulamalara bağlı olarak, P, K, Na ve Fe içeriği bakımından farklılık ortaya çıkmıştır. Sonuç: Sürgün uzunluğu ve meyve özellikleri bakımından A506 ve C9/1 bakteri uygulamalarının kontrole göre etkili olduğu görülmüştür. Kompost çayı uygulamasında besin elementi içeriği en yüksek değere ulaşmıştır. Tüm uygulamalar verimi olumlu yönde etkilemiştir.

BIOAVAILABILITY OF IRON TO PSEUDOMONAS FLUORESCENS STRAIN A506 ON FLOWERS OF PEAR

Pseudomonas fluorescens strain A506 (A506) produces an antibiotic toxic to Erwinia amylovora in defined culture media containing at least 0.1 mM FeCl3. To estimate the relative availability of iron on flowers, A506 was transformed with an 'iron biosensor', which consisted of an iron-regulated promoter (pvd) fused to an ice nucleation reporter gene (inaZ). A506 (pvd-inaZ) established high populations on blossoms, ranging from 105 to 107 colony forming units on pear. In two trials on screenhouse grown pear trees, A506 (pvd-inaZ) expressed high ice nucleation activity on blossoms indicating limited iron bioavailability, or a low-iron environment unlikely to induce antibiosis by A506. A506 (pvd-inaZ) also colonized blossoms when mixed with the iron chelate, ferric ethylenediaminedi-(o-hydroxyphenylacetic) acid (FeEDDHA). Co-treatment of flowers with a mixture of A506 (pvd-inaZ) and 3 mM FeEDDHA significantly decreased ice nucleation activity compared to flowers treated with A506 (pvd-inaZ) in water. Lower concentrations (i.e. 0.3 mM FeEDDHA) did not consistently increase iron available to A506 on flowers sufficient to suppress expression of the iron regulated reporter gene construct. These results indicate that pear flowers represent an iron-limited environment to A506 and that co-treatment with at least 3 mM FeEDDHA significantly increases the level of iron biologically available to this bacterium.

Management of Xanthomonas Leaf Blight of Onion with a Plant Activator, Biological Control Agents, and Copper Bactericides.

Xanthomonas leaf blight (Xanthomonas axonopodis pv. allii) is a yield-limiting disease of onion (Allium cepa) in the western United States. Frequent applications of copper-based bactericides amended with an ethylenebisdithiocarbamate fungicide (e.g., maneb or mancozeb, class B2 carcinogens) provide some disease suppression, but strategies to reduce conventional bactericide use are needed to minimize grower costs, environmental impact, and public exposure to class B2 pesticides. Applications of acibenzolar-S-methyl reduced in planta and epiphytic populations of X. axonopodis pv. allii as effectively as applications of copper hydroxide-mancozeb in growth chamber studies. Under field conditions, four weekly applications of acibenzolar-S-methyl reduced severity of Xanthomonas leaf blight as or more effectively than 9 to 12 weekly applications of copper hydroxide or copper hydroxide-mancozeb. Acibenzolar-S-methyl applications did not increase bulb yield or grade compared with copper bactericide treatments. However, bulb yield was reduced 22 to 27% when 10 weekly applications of acibenzolar-S-methyl were made in the absence of disease. Application of a commercial formulation of both Pantoea agglomerans strain C9-1 and Pseudomonas fluorescens strain A506 reduced severity of Xanthomonas leaf blight in field experiments. Weekly copper hydroxide applications starting 1 to 2 weeks before bulb initiation were as effective as weekly applications started 3 to 4 weeks before bulb initiation, irrespective of the maneb rate used. Integration of acibenzolar-S-methyl and biological control agents with copper hydroxide in a carefully timed spray program may eliminate the use of the class B2 carcinogens maneb and mancozeb on onion without compromising efficacy for management of Xanthomonas leaf blight.

An Extracellular Protease of Pseudomonas fluorescens Inactivates Antibiotics of Pantoea agglomerans

ABSTRACT Pseudomonas fluorescens A506 and Pantoea agglomerans strains Eh252 and C9-1 are biological control agents that suppress fire blight, an important disease of pear and apple caused by the bacterium Erwinia amylovora. Pseudomonas fluorescens strain A506 suppresses disease largely through competitive exclusion of E. amylovora on surfaces of blossoms, the primary infection court, whereas Pantoea agglomerans strains Eh252 and C9-1 produce antibiotics that are toxic to E. amylovora. In this study, an extracellular protease produced by A506 is characterized and evaluated for its capacity to inactivate the antibiotics produced by the strains of Pantoea agglomerans. Activity of the extracellular protease was optimal at pH 9 and inhibited by zinc- or calcium-chelators, indicating that the protease is an alkaline metalloprotease. In an agar plate bioassay, partially purified extracellular protease inactivated the antibiotics mccEh252 and herbicolin O, which are produced by Pantoea agglomerans strains Eh252 and C9-1, respectively. Derivatives of A506 deficient in extracellular protease production were obtained by transposon mutagenesis, and the aprX gene encoding the protease was cloned and sequenced. Strain A506 inactivated mccEh252 and herbicolin O in agar plate bioassays, whereas the aprX mutant did not inactivate the antibiotics. Both A506 and the aprX mutant were insensitive to antibiosis by C9-1 and Eh252; thus, the protease was not required to protect A506 from antibiosis. These data highlight a previously unknown role of the extracellular protease produced by Pseudomonas fluorescens A506 in interactions among plant-associated microbes.

Temperature and Pomaceous Flower Age Related to Colonization byErwinia amylovoraand Antagonists

ABSTRACT Fire blight of apple and pear is initiated by epiphytic populations of Erwinia amylovora on flower stigmas. Predicting this disease and managing it with microbial antagonists depends on an understanding of bacterial colonization on stigmas. Detached 'Manchurian' crab apple flowers were inoculated with E. amylovora and subjected to a range of constant temperatures or various fluctuating temperature regimes. Results may have application to disease risk assessment systems such as the Cougarblight model, which now are based on in vitro growth of the pathogen. In other experiments, detached crab apple flowers and attached 'Gala' apple flowers were maintained at different temperatures for various periods before inoculation with E. amylovora or antagonists (Pseudomonas fluorescens strain A506 and Pantoea agglomerans strains C9-1 and E325). Maximum stigma age supporting bacterial multiplication decreased as temperature increased, and was reduced by pollination. Stigmas were receptive to bacteria at ages older than previously reported, probably due to less interference from indigenous organisms. The study revealed antagonist limitations that possibly affect field performance (e.g., the inability of strain A506 to grow on relatively old stigmas conducive to the pathogen). Such deficiencies could be overcome by selecting other antagonists or using antagonist mixtures in the orchard.

Temporal Dynamics of the Biocontrol Agent Pseudomonas fluorescens Strain A506 in Flowers in Inoculated Pear Trees

ABSTRACT The colonization of individual flowers in mature pear orchards by Pseudomonas fluorescens strain A506 applied at different times during bloom was measured to determine the receptivity of flowers to colonization and the extent of intra-tree movement over time. Strain A506 populations in flowers open at inoculation were initially about 10(4) cells per flower and increased to approximately 10(6) cells per flower in flowers that were inoculated within about 5 days of opening. However, eventual populations decreased with further increases in flower age at inoculation to as few as about 10(3) cells per flower when inoculated flowers were more than 10 days old. Populations of strain A506 on flowers that opened after inoculation was initially very low at the time of petal expansion (<100 cells per flower) but increased rapidly with time after flower opening. The maximum population of strain A506 that developed on such flowers decreased with increasing time between inoculation and petal expansion; 10(4) to 10(5) cells of strain A506 eventually colonized flowers that opened within 7 days of inoculation, whereas fewer than 100 cells colonized flowers that opened 24 days or more after inoculation. Large total bacterial populations on A506-treated trees were associated with significant reductions in populations of Erwinia amylovora and reduced incidence of fire blight and severity of fruit russet.

Biological Control Agents for Fire Blight of Apple Compared Under Conditions Limiting Natural Dispersal.

The efficacy of Pantoea agglomerans strain E325 for control of fire blight of apple was determined in comparative field trials involving other bacterial antagonists. Concurrently, the importance of the natural dispersal of bacteria as a complicating factor was assessed. Tests were performed under two sets of conditions, those that allowed for the dispersal of bacteria via honeybee activity and those that minimized it through the use of translucent polyethylene enclosures around single trees. The enclosures also raised daytime temperatures and allowed for controlled wetting, two factors important to the development of blossom blight. Singleantagonist treatments with strain E325, Pseudomonas fluorescens strain A506, and P. agglomerans strain C9-1 were applied to open blossoms on each of 10 enclosed trees and 10 nonenclosed trees. During bloom, suspensions of antagonists (108 CFU/ml) were applied twice with a brush, and a suspension of Erwinia amylovora (107 CFU/ml) was subsequently applied once using the same method. Two days after inoculation with the pathogen, trees were misted to simulate precipitation. Flower-to-flower spread of antagonistic bacteria was less frequent on trees surrounded by plastic enclosures than on nonenclosed trees. The range and statistical separation of means for population size of E. amylovora and disease incidence among treatments were greater for enclosed trees than for nonenclosed trees. Based on these results, the natural spread of antagonists being compared may mask differences in their efficacy as biocontrol agents. Such distortions and resulting misinterpretations could be lessened by separating treatments widely in large orchard blocks and by monitoring microbial populations. Strain E325 from fresh or lyophilized cultures was consistently more effective than standard antagonists in suppressing E. amylovora and reducing disease incidence. To fully assess its potential use for fire blight, larger-scale trials under various conditions will be necessary.

Development and characterization of a green fluorescent protein-based bacterial biosensor for bioavailable toluene and related compounds.

A green fluorescent protein-based Pseudomonas fluorescens strain A506 biosensor was constructed and characterized for its potential to measure benzene, toluene, ethylbenzene, and related compounds in aqueous solutions. The biosensor is based on a plasmid carrying the toluene-benzene utilization (tbu) pathway transcriptional activator TbuT from Ralstonia pickettii PKO1 and a transcriptional fusion of its promoter PtbuA1 with a promoterless gfp gene on a broad-host-range promoter probe vector. TbuT was not limiting, since it was constitutively expressed by being fused to the neomycin phosphotransferase (nptII) promoter. The biosensor cells were readily induced, and fluorescence emission after induction periods of 3 h correlated well with toluene, benzene, ethylbenzene, and trichloroethylene concentrations. Our experiments using flow cytometry show that intermediate levels of gfp expression in response to toluene reflect uniform induction of cells. As the toluene concentration increases, the level of gfp expression per cell increases until saturation kinetics of the TbuT-PtbuA1 system are observed. Each inducer had a unique minimum concentration that was necessary for induction, with K(app) values that ranged from 3.3 +/- 1.8 microM for toluene to 35.6 +/- 16.6 microM for trichloroethylene (means +/- standard errors of the means), and maximal fluorescence response. The fluorescence response was specific for alkyl-substituted benzene derivatives and branched alkenes (di- and trichloroethylene, 2-methyl-2-butene). The biosensor responded in an additive fashion to the presence of multiple inducers and was unaffected by the presence of compounds that were not inducers, such as those present in gasoline. Flow cytometry revealed that, in response to toxic concentrations of gasoline, there was a small uninduced population and another larger fully induced population whose levels of fluorescence corresponded to the amount of effectors present in the sample. These results demonstrate the potential for green fluorescent protein-based bacterial biosensors to measure environmental contaminants.

Occurrence of Indole-3-Acetic Acid-Producing Bacteria on Pear Trees and Their Association with Fruit Russet

ABSTRACT A relatively high percentage of epiphytic bacteria on pear leaf and fruit surfaces had the ability to produce indole-3-acetic acid (IAA) in culture media supplemented with tryptophan. While over 50% of the strains produced at least small amounts of IAA in culture, about 25% of the strains exhibited high IAA production as evidenced by both colorimetric and high-performance liquid chromatography analysis of culture supernatants. A majority of the strains that produced high amounts of IAA were identified as Erwinia herbicola (Pantoea agglomerans), while some strains of Pseudomonas syringae, Pseudomonas viridiflava, Pseudomonas fluorescens, Pseudomonas putida, and Rahnella aquaticus that produced high amounts of IAA also were found on pear. Fruit russeting was significantly increased in 39 out of 46 trials over an 8-year period in which IAA-producing bacteria were applied to trees compared with control trees. A linear relationship was observed between fruit russet severity and the logarithm of the population size of different IAA-producing bacteria on trees in the 30 days after inoculation, when normalized for the amount of IAA produced by each strain in culture. On average, the severity of fruit russet was only about 77% that on control trees when trees were treated at the time of bloom with Pseudomonas fluorescens strain A506, which does not produce IAA. Both total bacterial populations on pear in the 30-day period following full bloom and fruit russet severity varied greatly from year to year and in different commercial orchards over a 10-year period. There was a strong linear correlation between the logarithm of total bacterial population sizes and fruit russet severity.

Crab Apple Blossoms as a Model for Research on Biological Control of Fire Blight

ABSTRACT Nonseasonal availability of pomaceous flowers could improve laboratory detection and prefield testing of biocontrol agents for fire blight of pear and apple. Crab apple was selected as a model because of its high flower productivity on 1-year-old wood, high susceptibility to fire blight, and availability from nurseries. Cultivars Manchurian and Snowdrift were manipulated to bloom once by transferring dormant nursery trees from a cold room to a greenhouse and a second time by defoliating trees and applying 1% cytokinin and 0.1% gibberellins to the buds with a brush. Different sets of trees were induced at different times to bloom, so that flowers were produced 12 months in the year. When known bacterial antagonists (Erwinia herbicola strain C9-1 and Pseudomonas fluorescens strain A506) were applied alone or in combination to the stigmas of detached crab apple blossoms prior to inoculation with the pathogen (E. amylovora strain Ea153), population interactions over time were comparable to those reported in previous studies involving pear or apple. In a subsequent series of experiments, the relative effects of 12 bacterial strains on stigmatic populations of strain Ea153 were similar for detached blossoms of crab apple in the laboratory, blossoms of intact crab apple trees in the greenhouse, and blossoms of pear and apple in the field. Additionally, when stigmas of detached crab apple blossoms were inoculated with antagonists (strains C9-1 and A506) and the pathogen, and later subjected to a 24-h wetting period, bacterial populations in the flower hypanthium increased and disease was suppressed. These studies indicate that crab apple blossoms can serve as a suitable model for year-round evaluation and study of biocontrol agents for fire blight.

Compatibility of Bacterial Antagonists ofErwinia amylovorawith Antibiotics Used to Control Fire Blight

In field experiments, two bacterial antagonists that suppress fire blight, Pseudomonas fluorescens strain A506 and a streptomycin-resistant mutant of Erwinia herbicola strain C9-1 (C9-lS), were sprayed onto apple blossoms at 30% bloom. Water, streptomycin sulfate, or oxytetracycline were sprayed onto blossoms 2 and 7 days after the antagonist applications to determine the effect of these chemicals on the population dynamics of P. fluorescens strain A506 and E. herbicola strain C9-1S during bloom. Incidences of recovery (the proportion of blossoms supporting detectable bacterial populations) and population sizes of P. fluorescens strain A506 and E. herbicola strain C9-1S on stigmas within individual blossoms were estimated with a dilution plating assay before and after each antibiotic application. Maximum incidences of recovery of P. fluorescens strain A506 and E. herbicola strain C9-1S from blossoms treated subsequently with water ranged from 58 to 100% and 47 to 100%, respectively; average population sizes of both strains were 10 4 to 10 6 CFU/flower. Streptomycin did not reduce the incidence of recovery or the population size of either antagonist. Oxytetracycline applications made 2 and 7 days after the antagonist applications reduced the incidence of recovery by 23 to 58% and also reduced the population size of both P. fluorescens strain A506 and E. herbicola strain C9-1S by 10- to 100-fold. In contrast, when the first oxytetracycline treatment was delayed to 7 days after the application of the antagonists, only a slight reduction in the incidence of recovery and the population size of either antagonist was observed. The population dynamics of P. fluorescens strain A506 and E. herbicola strain C9-1S, and presumably the degree of protection that they provide, need not be adversely affected by the concomitant usage of chemical antibiotics within the same season. Optimal integration of biological and chemical methods for suppression of fire blight, however, may require that oxytetracycline applications be delayed until after epiphytic populations of antagonists have become established on flowers.

Interactions of Antibiotics withPseudomonas fluorescensStrain A506 in the Control of Fire Blight and Frost Injury to Pear

Pseudomonas fluorescens strain A506 and the antibiotics streptomycin and oxytetracycline, to which this antagonist is resistant, were evaluated individually and in combination in field trials over a 16-year period to assess the interactions and relative efficacy of bactericides and antagonistic bacteria in the control of fire blight and frost injury to pear. Strain A506 maintained population sizes of greater than about 10 5 cells per fruiting spur for over 30 days on trees inoculated once with this strain. Over 90% of the flowers on inoculated trees harbored detectable populations of strain A506. The population sizes of strain A506 were as high on inoculated trees, to which streptomycin was applied, as on trees sprayed only with strain A506. The population sizes of ice nucleation active bacteria were about 100-fold lower on trees treated with strain A506 than on untreated control trees or trees sprayed only with antibiotics. The incidence of frost injury to pear fruit in natural frosts and of fire blight was reduced to about 40 and 50%, respectively, of that on untreated trees. The incidence of frost damage and fire blight was lower on trees treated both with strain A506 and antibiotics than with either agent alone. Strain A506 and antibiotics acted additively in the control of frost and disease.

INTERACTIONS BETWEEN THE BIOLOGICAL CONTROL AGENT PSEUDOMONAS FLUORESCENS A506 AND ERWINIA AMYLOVORA IN PEAR BLOSSOMS

In the greenhouse, Pseudomonas fluorescens strain A506 effectively colonized the pistils of pear blossoms. P. fluorescens A506 significantly reduced colonization of pear pistils by Erwinia amylovora when the biological control agent was inoculated 72 h in advance of the pathogen, but not when it was coinoculated with E. amylovora. P. fluorescens A506 probably excluded E. amylovora by preemptively utilizing a growth limiting resource required by the pathogen. A506 also colonized the nectaries of pear blossoms, in which it maintained high populations for 60-72 h []

Effect of Antagonistic Bacteria on Establishment of Honey Bee-DispersedErwinia amylovorain Pear Blossoms and on Fire Blight Control

In field trials conducted in 1991 and 1992 at Medford, OR, and in 1992 at Wenatchee, WA, Pseudomonas fluorescens strain A506 and Erwinia herbicola strain C9-1 established epiphytic populations on pear blossoms and were effective antagonists for the biological control of fire blight. Both bacterial antagonists, water, or streptomycin sulfate were applied to trees at 30% and full bloom. Pear trees were challenged inoculated with freeze-dried cells of E. amylovora vectored to blossoms by honey bees. One week after full bloom, the antagonists were established in more than 95% of treated blossoms in Oregon in 1991 and Washington in 1992, but in less than 50% of blossoms in Oregon in 1992. At the same bloom stage, 41% (Oregon, 1991), 27% (Oregon, 1992), and 49% (Washington, 1992) of water-treated blossoms had detectable populations of E. amylovora, whereas trees treated with bacterial antagonists always had a significantly lower (p 0.05) percentage of blossoms with detectable E. amylovora populations: 18-20% (Oregon, 1991), 9-15% (Oregon, 1992), and 8-17% (Washington, 1992). In Oregon in 1991, only 4% of blossoms treated with bacterial antagonists supported populations of E. amylovora that exceeded 10(5) cfu per blossom compared with 19% of blossoms treated with water; however, suppression of population size of E. amylovora by bacterial antagonists was not apparent in 1992. In 1991, fire blight symptoms developed in 8, 0.1, and 1% of blossom clusters treated with water, streptomycin, or bacterial antagonists, respectively. In 1992, the percentage of diseased blossom clusters in these same treatments in Oregon averaged 44, 2, and 22%, respectively, and 9, 2.5, and 4%, respectively, in Washington

Interactions Between the Biological Control AgentPseudomonas fluorescensA506 andErwinia amylovorain Pear Blossoms

In the greenhouse, Pseudomonas fluorescens strain A506 effectively colonized the pistils of pear blossoms. P. fluorescens A506 significantly reduced colonization of pear pistils by Erwinia amylovora when the biological control agent was inoculated 72 h in advance of the pathogen, but not when it was coinoculated with E. amylovora. P. fluorescens A506 probably excluded E. amylovora by preemptively utilizing a growth limiting resource required by the pathogen. A506 also colonized the nectaries of pear blossoms, in which it maintained high populations for 60-72 h []