INOCULATIONS WITH BACTERIA CAUSING PLANT DISEASE x.^-S 



fertilized parents is often available. When this is not sufficient, one 

 can commonly find or develop experimental units all genetically 

 identical through vegetative propagation. With such material any 

 variations secured can be studied without concern that the host may 

 have been obscuring pathogenicity. (7) Certain plant materials 

 can be cultivated in vitro on media containing only nutrients for 

 which the chemical formulae are known (reviewed by White, 1943). 



Simple Representative Inoculation Methods 



The actual method of making inoculations varies with circum- 

 stances. Some simpler methods are considered briefly by way of 

 illustration. Methods for testing the relative efficiency of several 

 technics are considered in a later section. 



SOIL "inoculation" 



The introduction of large numbers of pathogenic bacteria into the 

 soil depends upon growing sufficient quantities in cultures, either on 

 agar or in liquid media. Special flasks, bottles, and other containers 

 having adequate flat surfaces are employed. Most of the plant patho- 

 gens are aerobic and need to be incubated under pronounced aerobic 

 conditions if the best growth is to be secured. When agar is used the 

 surface growth on a suitable medium is washed or scraped off after 

 sufficient growth has appeared, and a suspension is made. When a 

 liquid medium is employed, a greater bacterial count per cubic centi- 

 meter is secured, with an organism like Phytomonas tumefaciens 

 (Smith and Town.) Bergey et al., by use of a medium less than 2 cm. 

 deep or well aerated by some other means. Satisfactory aeration may 

 be secured in deep liquid cultures by bubbling sterile air through a 

 scintered glass or other aerator placed in the medium. In large 

 containers aeration can be improved by a few pounds of pressure 

 which forces more air to dissolve in the liquid. Maintaining such 

 pressure also reduces contamination from leaky valves. Chemicals 

 that poise the oxidation-reduction potential may be helpful. The 

 highest count of active bacterial cells may be reached somewhat 

 before the maximum turbidity is attained. Considerable turbidity 

 is caused by bacterial gum. Usually the whole culture is employed 

 for soil treatment; but one should avoid adding too much extraneous 

 matter with the inoculum. Such aerated liquid cultures also work 

 well with some fungi. 



Soil may be "inoculated" by pouring liquid suspensions on rela- 

 tively dry soil, allowing the water to be absorbed long enough to 

 avoid puddling, and mixing. The quantity of culture used for each 

 plant varies. One might begin with 1 part of culture to 10 parts of 

 soil and use a handful of this mixture about the roots of each plant. 



Inoculations through the soil are considerably more difficult than 

 those with various other methods. 



SEED inoculation 



Perhaps the easiest way to infect a large population is through 

 treatment of the seed. Legume root nodule bacteria from a fresh, 



