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freezing behaviour and relative importance of heterogeneous ice nuclei in affecting supercooling of Citrus sinensis fruits were studied. The size of an ice nucleation active (INA) Pseudomonas syringae pool inhabiting fruits was positively correlated with the nucleation temperature (NT) of the plant tissue, with a mean of 3-69 log colony forming units (CFU) fruit"1 at NT~$> —2-5 °C. The INA bacterial pool was responsible for «23% of the nucleation events occurring at this temperature, and «29% were attributed to an additional nucleating source. The latter was sensitive to bacterial ice nucleation inhibitors, it occupied a different microniche from that of P. syringae nuclei, yet was neither a fungus nor any of the bacterial strains ever reported as active. Treatment with an ice nucleation inactive bacterium antagonistic to INA bacteria, a lentil lectin, a protease, and guanidine reduced mean nucleation temperatures (MNT) of fruits to ^ —2-58, —2-66, —4-21, and —4-52 °C, respectively, compared to a MNT^; — 1-67 CC for the controls. Thus, the citrus-associated nucleator apparently contained active proteinaceous components but was void of carbohydrate-like groups reportedly encountered at or near the bacterial ice nucleating site. Despite the different origins of citrus nuclei, bacterial nucleation inhibitors reduced nucleation in the field by 50% at NT^ —2-5 °C, an effect probably exerted through the proteinaceous site possessed by both nuclei.

The frost sensitivity of Citrus sinensis in relation to the presence of biogenic ice nuclei was studied. In commercially managed citrus groves the ice nucleation active (INA) bacterium Pseudomonas syringae reached 6 × 104 colony forming units (CFU) leaf−1, a population sufficiently high to catalyze ice formation. However, a transient loss of bacterial nucleation activity was noticeable at subzero field temperatures, followed by resumption as temperatures rose. This loss was apparently due to a temporary transition of INA to ice nucleation inactive (INI) bacteria. Field application of Bordeaux mixture, copper hydroxide, streptomycin, and 2-hydroxypropylmethanethiolsulfonate (HPMTS), resulted in reduction of INA bacterial populations to detectability (≤ 102 CFU leaf−1) limits. However, the corresponding reduction in ice nucleation events in treated samples as compared to controls at nucleation temperature ≥−3°C was not as dramatic. It ranged from approximately 7% in samples treated with the bactericide HPMTS, to 35% in samples treated with chemicals possessing combined bactericidal - fungicidal action (coppers). Since a quantitative relationship exists between ice nucleation events on individual leaves and the INA bacterial populations harbored by these leaves, these results suggest the co-existence of a bacterial and a proteinaceous, yet non-bacterial ice nucleating source in citrus, both active at ≥−3°C.

The total amount of copper, the amount of soluble but complexed copper, and the concentration of free Cu2+ ions on the surface of navel orange and bean leaves treated with different amounts of Cu(OH)2 or Bordeaux mixture were determined under field conditions. Total copper deposits decreased with time after spray application with apparent first-order kinetics with a half-life of approximately 45 and 35 days on navel orange and bean leaves, respectively. The concentration of soluble but complexed copper, however, increased for 20-30 days following spray application and comprised approximately 25 and 10% of the total copper on treated navel orange and bean leaves, respectively. The concentration of free Cu2+ ions on Cu(OH)2-treated leaves increased with time after spray application to a maximum concentration of approximately 100 ppb after about 10-20 days following treatment. No cells of copper-sensitive strains of Pseudomonas syringae survived after application to bean or navel orange leaves containing more than about 50 ppb of Cu2+. In constant, 10% or more of the cells of copper-tolerant strains survived on leaves containing up to 100 ppb of free Cu2+, and nearly all of the cells of tolerant strains survived on leaves containing lesser amounts of free Cu2+. Strains of P. syringae survived only at Cu2+ concentrations on leaves at or below the LC50 for Cu2+ for these strains determined in distilled water in vitro. Thus, even though very low concentrations of Cu2+ are present on leaves, coopper-sensitive strains are killed by the concentrations of free Cu2+ present on leaves. However, the concentration of free Cu2+ on leaves is only slightly less than that tolerated by copper-tolerant strains of P. syringae in vitro. Soluble but complexed forms of copper, while abundant on leaves, have no significant toxicity towards strains of P. syringae