Heterogeneous ice nuclei are necessary, and the common epiphytic ice nucleation active (INA) bacteria Pseudomonas syringae van Hall and Erwinia herbicola (Lihnis) Dye are sufficient to incite frost injury to sensitive plants at -5C. The ice nucleation activity of the bacteria occurs at the same temperatures at which frost injury to sensitive plants occurs in nature. Bacterial ice nucleation on leaves can be detected at about -2°C, whereas the leaves themselves, ie. without INA bacteria, contain nuclei active only at much lower temperatures. The temperature at which injury to plants occurs is predictable on the basis of the ice nucleation activity of leaf discs, which in turn depends on the number and ice nucleation activity of their resident bacteria. Bacterial isolates which are able to incite injury to corn at -5°C are always active as ice nuclei at -5°C. INA bacteria incited frost injury to all of the species of sensitive plants tested.

A replica plating method for rapid quantitation of ice nucleation-active (INA) bacteria was developed. Leaf washings of plant samples from California, Colorado, Florida, Louisiana, and Wisconsin were tested for the presence of INA bacteria. Of the 95 plant species sampled, 74 were found to harbor INA bacteria. Only the conifers were, as a group, unlikely to harbor INA bacteria. All of the INA bacteria isolated resembled either Pseudomonas syringae or Erwinia herbicola. Sufficient numbers of INA bacteria were present on the samples to account for the ice nuclei associated with leaves that are necessary for freezing injury to occur. Numbers of INA bacteria were large enough to suggest that plant surfaces may constitute a significant source of atmospheric ice nuclei.

No abstract

Not every cell of a given bacterial isolate that has ice-nucleating properties can serve as an ice nucleus at any given time and temperature. The ratio between the number of ice nuclei and number of bacterial cells in a culture (i.e. nucleation frequency) was found to vary with incubation temperature, growth medium composition, culture age, and genotype. Optimal conditions for ice nucleus production in vitro included incubation of the bacterial cells at 20 to 24°C on nutrient agar containing glycerol. The relationship between nucleation frequency and frost injury was examined by subjectiag corn seedlngs to -4°C immediately after they were sprayed with bacterial suspensions with different nucleation frequencies and by following both ice nucleus concentration and bacterial population size on leaves of corn seedllngs as a function of time after bacterial application. (1, 4). The other E. herbicola isolates (Nos. 1, 2, 10, 24A, and 30B) were isolated from dilution plates of leaf washings of field-grown corn plants. P. syringae isolate Y-26 and P. syringae pv. coronafaciens isolate No. 5 were from the culture collection of A. Kelman, University of Wisconsin-Madison.Bacterial cultures were grown on NA (3.3 g Bacto peptone, 2.7 g Difco nutrient broth, 2.0 g yeast extract, and 15.0 g Bacto agar/ L distilled H20); NA containing 2.5% glycerol (v/v) or 2.5% glucose (w/v); King's medium B (2); nutrient broth containing 2.5% glycerol, minimal broth (1.0 g glucose, 7.0 g K2HPO4, 0.5 g sodium citrate, 0.1 g MgSO4.7H20, and 1.0 g (NH4)2SO4 in 1 L distilled H20) and minimal agar (minimal broth + 15.0 g Bacto agar/L), at the temperatures and for the times specified for each experiment.4 Liquid cultures were agitated with a reciprocal shaker during incubation.Ice Nucleation Activity of Bacterial Suspensions. Bacterial suspensions for ice nucleus concentration determinations were prepared from colonies on agar medium. Cells were removed with a loop and suspended in either distilled H20 or 0.1 M phosphate buffer (pH 7.0). Broth cultures were diluted directly with distilled H20. All dilutions of cultures were 100-fold or greater in the suspensions in which ice nucleus concentrations were measured. Viable cell densities were determined by dilution plating. Ice nucleation spectra were determined as described elsewhere (4,8

No abstract