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Michele R. Warmund and James T. English

INA bacteria were isolated from primary flowers of `Totem' strawberry (Fragaria ×ananassa Duch.) plants that had been previously inoculated with strain Cit 7 of Pseudomonas syringae van Hall or noninoculated to determine their relationship to ice-nucleation temperature and floral injury. Mean ice-nucleation temperature of inoculated and noninoculated flowers was -2.2 and -2.8 °C, respectively. Primary flowers of noninoculated plants survived lower temperatures than those of inoculated plants. In another experiment, noninoculated plants were misted with sterile deionized water and incubated for 0, 12, 24, 36, or 48 hours at 25 °C day/10 °C night, and naturally occurring INA bacteria were isolated from primary flowers. INA bacterial densities increased exponentially with increasing incubation period. The critical wetness period for INA bacteria to establish a sufficient density to increase the likelihood of floral injury at -2.5 °C was 24 hours. Longer wetness periods resulted in higher INA bacterial densities but did not increase the floral mortality rate. Thermal analysis demonstrated that the ice nucleation temperature was associated with strawberry floral injury. Thus, low temperature survival of flowers was adversely affected by moisture for ≥24 h due to the presence of a sufficient density of INA bacteria to incite ice formation and floral injury.

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Jeffrey Anderson

Epiphytic populations of ice nucleation active (INA) bacteria responded differently to preconditioning temperature treatments depending on plant species. Ice nucleation activity of inoculated tomato (Lycopersicon esculentum Mill.), squash (Cucurbita pepo L.), and cucumber (Cucumis sativus L.) seedlings was not affected by preconditioning temperature treatments of 7, 21, or 33C for 3 hr prior to freezing assays. In contrast, preconditioning at 33C for 3 hr prior to assay decreased mean freezing temperatures of inoculated pine shoots compared with preconditioning at 5C. Preconditioning treatments of pine shoots had a greater effect on freezing temperatures when tissues were submerged in water during treatment. Cucumber seedlings responded similarly regardless of whether they were exposed to preconditioning treatments with dry surfaces or in a saturated environment. Preconditioning temperatures had a greater effect on ice nucleation activity of bacterial suspensions than on plants harboring INA bacteria.

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Michele R. Warmund and James T. English

Experiments were conducted to determine the temperatures at which different densities of INA bacteria incite ice crystallization on `Totem' strawberry flowers and to determine if there is a relationship between densities of INA bacteria on strawberry flowers and floral injury. Primary flowers were inoculated with Pseudomonas syringae at 106 cells/ml buffer, incubated at 25°C day/10°C night and 100% RH for 48 h, and exposed to –2.0°C. No ice nucleation occurred on these inoculated flowers and all of the flowers survived. However, when inoculated flowers were subjected to lower temperatures, ice nucleation occurred at –2.2°C and few of the flowers survived. In contrast, ice crystals formed on the surface of most non-inoculated flowers at –2.8°C and 21% of the flowers survived exposure to –3.5°C. When INA bacterial densities were ≈105 colony forming units/g dry wt, floral injury occurred at a warmer temperature than to flowers that had lower bacterial densities.

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Michele R. Warmund and James T. English

In 1993, ice-nucleation-active (INA) bacteria were isolated from `Redwing' red raspberries (Rubus idaeus L. var. idaeus) at five pigmentation stages. Fruit were also subjected to thermal analysis to determine the ice nucleation temperatures. INA bacteria were recovered from nearly all fruit samples, and the bacterial populations tended to decrease with greater red color development (i.e., fruit maturation). However, the ice nucleation temperature was not affected by the stage of fruit pigmentation. In 1994, INA bacterial densities were similar among fruit at the three pigmentation stages sampled. INA bacteria were recovered more often from the calyx rather than the drupe surface of these fruit. INA bacteria also were detected on pistils of some fruit. Red and pink fruit, which were nucleated with ice, had greater receptacle injury than mottled, yellow, or green fruit, but INA bacterial densities apparently were not related to injury. Thus, the injury response of fruit at different pigmentation (or development) stages indicated that nonbacterial ice nuclei may be involved in freezing injury of developing raspberries.

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Jeffrey A. Anderson and Julia Whitworth

Frostgard did not effectively promote the supercooling of flowering `Arking' strawberry (Fragaria ×ananassa Duch.) plants in the presence or absence of ice-nucleation-active bacteria when applied as a spray in laboratory experiments. Frostgard effectively promoted supercooling and reduced the ice propagation rate of aqueous solutions. Detached leaves infiltrated with Frostgard exhibited a negative linear relationship between freezing temperature and Frostgard concentrations from 0% to 20% (by volume). Leaves infiltrated with 20% Frostgard supercooled 1.7C lower than those infiltrated with distilled water. Ice propagation barriers in strawberry plants were observed. Individual leaves froze independently, and a thermal ice propagation barrier sometimes was observed at the crown.

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Beth Ann A. Workmaster, Jiwan P. Palta, and Michael Wisniewski

Infrared video thermography was used to study formation of ice in leaves, stems, and fruit of cranberry (Vaccinium macrocarpon Ait. `Stevens'). Ice formed on the plant surface at -1 or -2 °C by freezing of a droplet of water containing ice nucleation-active bacteria (Pseudomonas syringae van Hall). Samples were then cooled to a minimum of -8 °C. Observations on the initiation and propagation of ice were recorded. Leaves froze only when ice was present on the abaxial surface. Once initiated, ice propagated to the stem and then readily to other leaves. In both unripe and ripe fruit, ice propagation from the stem to the fruit via the pedicel was not observed. Fruit remained supercooled for up to 1 hour after ice was present in the stem. Fruit could only be nucleated when ice was present at the calyx (distal) end. Red (ripe) berries supercooled to colder temperatures and for longer durations than blush (unripe) berries before an apparent intrinsic nucleation event occurred. These observations provide evidence that leaves are nucleated by ice penetration via stomata. The ability of fruit to supercool appears to be related to the presence of barriers to extrinsic ice propagation at both the pedicel and fruit surface. Stomata at the calyx end of the fruit in the remnant nectary area may provide avenues for extrinsic ice nucleation.

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Michele R. Warmund and James T. English

Cryoprotectants were applied at labeled rates to primary flowers of `Honeoye' strawberry (Fragaria × ananassa Duch.) plants at full bloom to determine their effects on the floral organs. Frostgard at 50 ml/liter or KDL at 22 ml/liter injured pistils and resulted in misshapened fruit. Floral buds that were closed when cryoprotectants were applied were uninjured. In other experiments, efficacies of cryoprotectants were determined after floral tissues of `Honeoye' strawberry plants were inoculated or not inoculated with the ice-nucleation-active (INA) bacteria, Pseudomonas syringae van Hall and subjected to sub-freezing temperatures. None of the products protected primary or secondary flowers against freezing injury regardless of the occurrence of INA bacteria. INA bacteria were not recovered from primary flowers of treated plants that were killed by low temperature exposure, indicating that non-bacterial nuclei may incite freezing in these tissues.

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Beth Ann A. Workmaster, Michael Wisniewski, and Jiwan P. Palta

Infrared video thermography has recently been used to visualize ice nucleation and propagation in plants. At the UW–Madison Biotron facility, we studied the formation of ice in various parts of fruit-bearing cranberry (Vaccinium macrocarpon Ait.) uprights. The fruits were at the blush to red stages of ripening. Samples were nucleated at –1 or –2°C with ice-nucleating-active bacteria (Pseudomonas syringae). Following nucleation, samples were cooled to –6°C in ≈1 hour. The following observations were made: 1) When nucleated at a cut end, ice propagated rapidly throughout the stem and into the leaves at a tissue temperature of about –4°C. However, ice did not propagate from the stem through the pedicel to reach the fruit. During the 1 hour after ice propagation in the stem, the fruit remained supercooled. 2) Within the duration of the experiment, leaves could not be nucleated from the upper surface. Ice from the lower leaf surface did nucleate the leaf, and ice propagated from the leaf to the stem and other leaves readily. 3) Both red and blush berries could only be nucleated at the calyx end of the fruit. 4) Red berries supercooled to colder temperatures and for longer durations than the blush berries. 5) In support of our previous studies, red berries were able to tolerate some ice in their tissue. These observations suggest that: 1) The upper leaf surface and the fruit surface (other than the calyx end) are barriers to ice propagation in the cranberry plant; and 2) at later stages of fruit ripening the pedicel becomes an ice nucleation barrier from the stem to the fruit. This may contribute to the ability of the cranberry fruit to supercool.

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Michael Wisniewski, Michael Glenn, and Mick Fuller

Most plants exhibit the ability to supercool to some extent without freezing. The extent of supercooling, however, is limited by the action of intrinsic and extrinsic ice nucleating agents which initiate ice formation and propagation within a plant at relatively warm subzero temperatures (-1.5 to -3.5 °C). In herbaceous plants, extrinsic ice-nucleating agents (such as ice-nucleation bacteria, dew, and other good nucleating agents) significantly limit the ability to supercool below 0 °C. It is believed that with an absence of these extrinsic nucleating agents that plants could supercool to less than -4 °C. Other evidence indicates that intrinsic nucleating agents may also significantly limit the extent of supercooling. Questions also exist about nucleation in woody plants and especially the new growth (flowers, leaves, and shoots) present in spring. A better understanding of how freezing is initiated in plants has been limited by the inability to determine and visualize the initial site of ice nucleation and pattern of ice propagation. We have used infrared video thermography to study freezing in young tomato (Lycopersicon esculentum) plants and to determine if a hydrophobic barrier on the plant surface could prevent the action of extrinsic nucleating agents such as Ice + bacterial strain (Cit7) of Pseudomonas syringae from initiating freezing within a plant. Tomato plants were grown in a greenhouse in individual pots and used when they were 4 to 6 weeks old. Freezing tests were conducted in a programmable freezing chamber, and freezing was visualized and recorded on videotape using an infrared radiometer. Freezing of the plants was extrinsically induced by the application of droplets (5 μl) of water containing Cit7. To provide a barrier to the action of extrinsic ice-nucleating agents, an emulsion of hydrophobic kaolin was applied to the plant surface before applying an extrinsic nucleating agent. Results indicate that dry, young tomato plants can supercool to as low as -6 °C whereas plants having a single droplet of Cit7 would freeze at -1.5 to -2.5 °C. Applying the hydrophobic barrier blocked the effect of Cit7 and allowed the plants to also supercool to -6 °C, despite the presence of frozen droplets. Experiments under natural freezing conditions are in progress.

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Michael Wisniewski, D. Michael Glenn, and Michael P. Fuller

Extrinsic ice nucleating agents (such as ice-nucleation-active bacteria, dew, etc.) significantly limit the ability of herbaceous plants to supercool. It is believed that with an absence of these extrinsic nucleating agents, a plant could supercool to less than -4 °C. Other evidence, however, indicates that intrinsic nucleating agents may limit the extent of supercooling. Infrared video thermography was used to study freezing in young, `Rutgers' tomato (Lycopersicon esculentum L.) plants and to determine if a hydrophobic barrier on the plant surface could prevent extrinsic nucleating agents such as Ice+ bacterial strain (Cit7) of Pseudomonas syringae Van Hall from initiating freezing within a plant. Freezing tests were conducted in a programmable freezing chamber, a radiative frost chamber, and outdoors. Freezing was visualized and recorded on videotape using an infrared radiometer. Freezing of the plants was induced extrinsically by application of droplets (5 to 7 μL) of water containing Cit7. To provide a barrier to the action of extrinsic ice nucleating agents, an emulsion of hydrophobic kaolin (aluminum silicate mineral) was applied to the plant surface before application of an extrinsic nucleating agent. Results indicate that dry, young tomato plants can supercool to as low as -6 °C whereas plants having a single droplet of Cit7 would freeze at -1.5 to -2.5 °C. Application of the hydrophobic barrier blocked the effect of Cit7 and allowed whole plants to also supercool to -6 °C, despite the presence of frozen droplets on the leaf surface. When whole plants were sprayed with water and Cit7 using an aerosol sprayer and exposed to -3 °C, plants coated with the hydrophobic particle film exhibited significantly less foliar injury then nontreated plants. Similar results were obtained using the radiative frost chamber. Experiments conducted under natural frost conditions also resulted in less injury to the coated plants. The hydrophobic kaolin particle film performed better at preventing plants from freezing due to extrinsic ice nucleation than nonaltered, hydrophyllic kaolin alone or an antitranspirant with putative frost protection properties.