High Ice Nucleation Activity Research Articles

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.

Bioavailability of Iron to Pseudomonas fluorescens Strain A506 on Flowers of Pear and Apple

ABSTRACT The addition of 0.1 mM FeCl(3) to a defined culture medium induces the bacterial epiphyte Pseudomonas fluorescens strain A506 (A506) to produce an antibiotic toxic to the fire blight pathogen, Erwinia amylovora. Consequently, because A506 is registered and applied as a commercial product to suppress E. amylovora before floral infection of pear and apple, the relative availability of iron to A506 on surfaces of pear and apple flowers is of potential significance. An 'iron biosensor' construct of A506 was developed by transformation with an iron-regulated promoter (pvd) fused to a promoterless ice nucleation reporter gene (inaZ). This construct, A506 (pvd-inaZ), established high populations on pear and apple flowers, ranging from 10(4) to 10(6) CFU/flower. In seven trials on pear and apple trees, A506 (pvd-inaZ) expressed high ice nucleation activity (INA) on flowers, indicating limited iron bioavailability or a low-iron environment unlikely to induce antibiotic production by A506. A506 (pvd-inaZ) also colonized flowers when mixed with chemicals containing iron: FeSO(4) or the iron chelates ferric ethylenediaminedi-(o-hydroxyphenyl-acetic) acid (FeEDDHA) and ferric diethylenetriamine pentaacetate (FeDTPA). These compounds represent an array of commercial iron formulations applied to foliage to avert iron chlorosis. Treatment of flowers with a mixture of A506 (pvd-inaZ) and 3 mM FeEDDHA or FeDTPA significantly decreased INA compared with flowers treated with A506 (pvd-inaZ) in water. Lower concentrations (0.3 mM) of FeEDDHA, however, did not consistently suppress INA. These results indicate that apple and pear flowers represent an iron-limited environment to A506 and that treatment with 3 mM FeEDDHA is needed to increase significantly the level of iron available to this bacterium.

Observations of Ice Nucleation and Propagation in Flowers of Japanese Pear (Pyrus pyrifolia Nakai) using Infrared Video Thermography

Ice formation and propagation in the flower and young fruit of Japanese pear 'Kosui' were investigated with infrared (IR) video thermography imaging system. The changes of thermal response (exotherm) in freezing events could be detected using the infrared thermography camera with exotherm rise of 2-5 °C accompanied by a marked color change in the tissues. With first bloom flowers, the temperature of peduncles decreased faster than that of the receptacle; the initial ice nucleation occurred primarily in petals, sepals and/or receptacle. Subsequently, ice was propagated along the peduncle. The temperature of receptacles was above that of peduncles during the freezing test at full bloom; the sepals and/or receptacle were initially frozen, then ice nucleation was extended to the petals along the peduncle. These results suggest that the sepals and/or receptacle where the initial nucleation occurred may be an intrinsic or extrinsic source of high ice nucleation activity in flowers of the Japanese pear 'Kosui'. In contrast with first bloom and full bloom stage, the exotherms during freezing could not be detected clearly in young fruits. However, cold injury appeared as a spotty stain on the skin of young fruits immediately upon being subjected to chilling.

Construction of ice nucleation active Enterobacter cloacae for control of insect pests

Ice nucleation active (INA) bacteria are the most potent heterogeneous ice nuclei in nature, which have become an important biological resource for diverse applications. Many researches have proved that INA bacteria can raise the supercooling points (SCPs) of insect pests, then reduce their cold hardiness. However, INA bacteria’s inefficient colonization on the surface or in the guts of insects, and the high incidence of frost injury induced by their release hampered the application of INA bacteria in controlling insect pests in agricultural fields. In this study, we constructed a recombinant plasmid mob-Tn5 -iceA with the ability of broad-host-range conjugal mobilization and integration of the ina gene of iceA into chromosomal DNA of many gram-negative bacteria by Tn5 transposition. In addition, Ent. cloacae strains stably carrying iceA and expressing high ice nucleation activity (INA), even in the absence of antibiotic pressure, were constructed through conjugal mobilization and Tn5 transposition. Ent. cloacae strains have been reported to be able to efficiently colonize in the guts of insects, but have weak plant epiphytic ability. Therefore, these transgenic Ent. cloacae may be promising candidates for control of insect pests in agricultural fields.

The operon for cytokinin biosynthesis of Erwinia herbicola pv. gypsophilae contains two promoters and is plant induced.

The operon for cytokinin biosynthesis in the gall-forming bacterium Erwinia herbicola pv. gypsophilae (Ehg) has been previously shown to reside on an indigenous plasmid (pPATH(Ehg)) that is mandatory for pathogenicity. This operon consists of two genes: the first open reading frame (pre-etz) is of unknown function, whereas the second one (etz) encodes for isopentenyl transferase. Northern hybridization performed with the wild-type strain Ehg824-1 grown in Luria-Bertani broth demonstrated two transcripts of which an etz-specific transcript (1.0 kb) was predominant. Fusion of upstream DNA fragments of both pre-etz and etz to the ice nucleation reporter gene inaZ in pVSP61 showed high ice nucleation activity in both cultures, confirming the presence of two independent promoters. An increase of 1-1.5 orders in transcriptional activity of these promoters was observed following inoculation of gypsophila cuttings. Mutants of Ehg824-1 were generated by insertion of inaZ into pre-etz and etz using the transposon reporter Tn3-Spice. An increase of about two orders in transcriptional activity was recorded with both mutants following inoculation of gypsophila or bean cuttings. A similar induction was also observed when the bacteria were applied to the leaf surface of these plants. Unlike other virulence genes present on the pPATH(Ehg), neither pre-etz nor etz was regulated by the adjacent hrp gene cluster.

Role of Ice Nucleation and Antifreeze Activities in Pathogenesis and Growth of Snow Molds

ABSTRACT We examined the ability of snow molds to grow at temperatures from -5 to 30 degrees C and to influence the growth of ice through assays for ice nucleation and antifreeze activities. Isolates of Coprinus psychromorbidus (low temperature basidiomycete variant), Microdochium nivale, Typhula phacorrhiza, T. ishikariensis, T. incarnata, and T. canadensis all grew at -5 degrees C, whereas Sclerotinia borealis and S. homoeocarpa did not grow at temperatures below 4 degrees C. The highest threshold ice nucleation temperature was -7 degrees C. Because snow molds are most damaging to their hosts at temperatures above this, our results imply that the pathogenesis of these fungi is not dependent on ice nucleation activity to cause freeze-wounding of host plants. All snow molds that grew at subzero temperatures also exhibited antifreeze activity in the growth medium and in the soluble and insoluble hyphal fractions, with the exception of M. nivale and one isolate of T. canadensis. The lack of high ice nucleation activity combined with the presence of antifreeze activity in all fungal fractions indicates that snow molds can moderate their environment to inhibit or modify intra- and extracellular ice formation, which helps explain their ability to grow at subzero temperatures under snow cover.

Ice-nucleating activity of Pseudomonas syringae cultivated on a natural substrate; influence of phosphate

A novel kind of culture medium is used for the production Pseudomonas syringae with a high ice-nucleating activity. It is based on a natural substrate, wheat bran, which contains a relatively high proportion of phytate. The double salt of phytic acid is the precursor of a major component of the ice-nucleating site (myo-inositol). Experiments with the purified components show the positive effects on the ice-nucleating activity. The use of the wheat bran medium seems to be specifically efficient on class A bacteria, which is the most active type of P. syringae. We have shown that inorganic phosphate starvation during the preculture of P. syringae leads to higher ice-nucleating activity.

Culture conditions of Erwinia uredovora in reference to its high ice-nucleating activity of the culture supernatant.

Culture conditions of Erwinia uredovora in reference to its high ice-nucleating activity of the culture supernatant.

Cultivation and properties of ice-nucleation active bacteriaPseudomonas syringae CCM 4073

An ice-nucleating bacteriumPseudomonassyringae CCM 4073 selected from 30 ice-nucleating bacteria was cultivated in pilot-plant bioreactors at 28°C; the cells showed a high ice-nucleation activity even in dry state, sublimation activity and activation after imposition into refrigenator.

Ice-nucleating activity of Pseudomonas fluorescens

Pseudomonas fluorescens KUIN-1 can cause the freezing of water at relatively warm termperatures (−2.9 to −30°C). Glucose, glycerol, and citric acid were good substrates yielding cells with high ice-nucleating activity. Ammonium salts were good nitrogen sourcfes yielding cells with high ice-nucleating activity. The ice-nucleating activity of KUIN-1 was not affected by pH between pH 5.5 to pH 8.0. The ice-nucleating activity of cells was greatly influenced by heat treatment.

Ice Nucleation and Supercooling in Freezesensitive Peach and Sweet Cherry Tissues

Abstract Ice nucleation temperatures were measured in peach [Prunus persica (L.) Batsch ‘Redhaven’] and sweet cherry (Prunus avium L. ‘Bing’) flowers, fruits, and stems by differential thermal analysis and by controlled freezing in test tubes and of droplets. The mean nucleation temperatures of peach and sweet cherry flowers were –4° to – 6°C, while those of 0.5-cm stem segments were –6° to –7°. The highest ice nucleation activity in peach flowers appeared to be associated with the pedicels. A logarithmic function described the relationship between stem weight and ice nucleation temperature, with 1.5-g (16-cm) stem segments freezing as high as –3°. Apparently, stem tissue contained a lower concentration of ice nucleators active above –5° than did the floral tissue. Homogenization of the tissues reduced their ice nucleation activity. Structural integrity may be necessary for the manifestation of optimal activity. Ice nucleation-active (INA) Pseudomonas syringae bacteria were isolated from the surfaces of peach and sweet cherry floral tissues only occasionally and then at low populations. The absence of epiphytic INA bacteria from these tissues suggested that ice nucleation was promoted by intrinsic ice nucleators within the plant. These intrinsic ice nucleators appeared to be active at temperatures similar to INA bacteria. Although these nucleators may be less numerous than bacterial ice nuclei on heavily colonized plant surfaces, their presence nonetheless suggests that control of INA bacteria or inhibition of the ice nucleation activity of INA bacteria alone may be insufficient to prevent ice nucleation and freezing injury of freeze-sensitive flowers and fruits during spring frosts in the orchard.

Frost Injury and Heterogeneous Ice Nucleation in Leaves of Tuber-Bearing Solanum Species

The heterogeneous ice nucleation characteristics and frost injury in supercooled leaves upon ice formation were studied in nonhardened and cold-hardened species and crosses of tuber-bearing Solanum. The ice nucleation activity of the leaves was low at temperatures just below 0 degrees C and further decreased as a result of cold acclimation. In the absence of supercooling, the nonhardened and cold-hardened leaves tolerated extracellular freezing between -3.5 degrees and -8.5 degrees C. However, if ice initiation in the supercooled leaves occurred at any temperature below -2.6 degrees C, the leaves were lethally injured.To prevent supercooling in these leaves, various nucleants were tested for their ice nucleating ability. One% aqueous suspensions of fluorophlogopite and acetoacetanilide were found to be effective in ice nucleation of the Solanum leaves above -1 degrees C. They had threshold temperatures of -0.7 degrees and -0.8 degrees C, respectively, for freezing in distilled H(2)O. Although freezing could be initiated in the Solanum leaves above -1 degrees C with both the nucleants, 1% aqueous fluorophlogopite suspension showed overall higher ice nucleation activity than acetoacetanilide and was nontoxic to the leaves. The cold-hardened leaves survived between -2.5 degrees and -6.5 degrees using 1% aqueous fluorophlogopite suspension as a nucleant. The killing temperatures in the cold-hardened leaves were similar to those determined using ice as a nucleant. However, in the nonhardened leaves, use of fluorophlogopite as a nucleant resulted in lethal injury at higher temperatures than those estimated using ice as a nucleant.

The Local Cooling Ice Nucleation Model

Abstract A local cooling ice nucleation model is proposed to explain the high ice nucleation activity of soluble compounds which have endothermic heats of solution. Partial dissolution of the nuclei particle is required to produce local cooling and high activity. The amount of local cooling is shown to be governed by the heat and mass transfer processes in the boundary layer immediately adjacent to a dissolving ice nucleus. Variations in the heat transfer processes can be predicted from changes in experimental conditions. The effects of various experimental conditions upon local cooling and ice nucleation activity are the general result.