25 
(6) Studies with genetically marked strains have shown that the bacteria 
do not survive well through a full yearly cycle, i.e., their 
populations drop to very low levels during the dry season. These 
epiphytic bacteria do not survive for more than a month in the 
soil. The bacteria can survive up to a year in plant debris on 
the surface of the soil, but die rapidly when plant debris is 
incorporated into the soil. All plant debris in trial locations 
will be promptly incorporated into the soil upon completion of 
each experiment. Although some movement of bacteria to aerial 
sites near treatment locations by insect or aerial dispersal is 
possible, the numbers of viable cells transported has been shown 
to be very small; and these cells are subject to biological and 
physical processes limiting survival. 
(7) Wild-type strains of both bacteria have been sprayed on the field 
over several years with no adverse effects on humans. syringae 
p.v. syringae is not a human pathogen; indeed, it does not grow at 
temperatures above 33-34 °C. Pathogenicity of Eh_ herbicola 
to humans appears to be a property of Eb_ herbicola strains of 
human origin but not of plant origin. The strains to be used in 
this study were isolated from healthy plants. 
(8) With the exception of streptomycin and tetracycline, antibiotics 
are not commonly used for plant disease control in temperate regions. 
Use of resistance markers to these antibiotics will be specifically 
avoided . 
(9) Growth chamber and greenhouse experiments will be performed to 
ascertain that the engineered strains do not cause frost injury to 
plants. 
(10) The impact of INA - deletion mutants on rainfall patterns is thought 
to be extremely small or non-existent. The extremely small scale 
of these trials compared to the amount of agricultural and natural 
vegetation suggest that any potential reductions of atmospheric 
ice nuclei would be negligible. There are no reports of alterations 
in rainfall patterns following large-scale use of agricultural 
bactericides over the last four decades. The microbiological 
impact of field trials on the natural epiphytic populations of 
ice nucleating bacteria would be negligible compared to that 
caused by standard agroncmic practices such as orchard pruning 
or crop selection by farmers. 
Dr. Vidaver said that "worst case" experiments have already been done, i.e., 
these organisms modified by classical genetic techniques have already been 
released. In addition, she said that the populations to be released are 
8 to 10 orders of magnitude lower then those normally found in the environment. 
Dr. Tolin felt the investigators had satisfactorily addressed the issues 
raised at the October 25, 1982, RAC meeting. Dr. Fedoroff said she was 
satisfied with the proposal. She said the investigators have admirably 
rnoi 
