Measurement of ice nucleation-active bacteria on plants and in precipitation by quantitative PCR

Ice nucleation-active (INA) bacteria may function as high-temperature ice-nucleating particles (INP) in clouds, but their effective contribution to atmospheric processes, i.e., their potential to trigger glaciation and precipitation, remains uncertain. We know little about their abundance on natural vegetation, factors that trigger their release, or persistence of their ice nucleation activity once airborne. To facilitate these investigations, we developed two quantitative PCR (qPCR) tests of the ina gene to directly count INA bacteria in environmental samples. Each of two primer pairs amplified most alleles of the ina gene and, taken together, they should amplify all known alleles. To aid primer design, we collected many new INA isolates. Alignment of their partial ina sequences revealed new and deeply branching clades, including sequences from Pseudomonas syringae pv. atropurpurea, Ps. viridiflava, Pantoea agglomerans, Xanthomonas campestris, and possibly Ps. putida, Ps. auricularis, and Ps. poae. qPCR of leaf washings recorded ∼10(8) ina genes g(-1) fresh weight of foliage on cereals and 10(5) to 10(7) g(-1) on broadleaf crops. Much lower populations were found on most naturally occurring vegetation. In fresh snow, ina genes from various INA bacteria were detected in about half the samples but at abundances that could have accounted for only a minor proportion of INP at -10°C (assuming one ina gene per INA bacterium). Despite this, an apparent biological source contributed an average of ∼85% of INP active at -10°C in snow samples. In contrast, a thunderstorm hail sample contained 0.3 INA bacteria per INP active at -10°C, suggesting a significant contribution to this sample.