2o8 Journal of Agricultural Research voi. xix. no. 5 



and accuracy of the work. A leaf was inoculated by wiping both under 

 and upper surface of the freshly punctured portion with a cotton swab 

 from a dilution dish, the swab being finally left on the upper surface. 

 The inoculated plant was wrapped in paraffin paper, which served to 

 retain moisture and to prevent accidental contamination from outside 

 sources. Other plants were inoculated with pure cultures of P. citri as 

 controls, and others were set up with the swabs merely wet with sterile 

 water. The series were held at least a week in glass inoculation cases 

 where conditions were near the optimum for canker development; later 

 they were removed to the greenhouse benches. The first observations 

 and records were made as a rule two to four weeks after inoculation. 

 Final records were deferred until four to eight weeks after inoculation 

 in order to insure the detection of any unusually slow development of 

 infection such as occurred when the inoculum contained only a few 

 organisms. The records show that between 90 and 95 per cent oi the in- 

 fections were apparent at the first observation and that no material 

 increase was secured by holding beyond the second observation. 



Variations of this method were tried out during its evolutionary 

 development and to some extent in routine work as special considerations 

 seemed to warrant. In many of the earlier series absorbent cotton 

 wicks from small bottles of sterile water were placed in contact with the 

 inoculation swabs on the leaves. This precaution to secure a prolonged 

 moist condition proved to be unnecessary. An inoculum of mud paste, 

 made by adding only a little water to the soil sample and applied w'ith a 

 backing of cloth or cotton as a sort of poultice over the punctured area, 

 gave distinctly fewer infections than the soil solution in much greater dilu- 

 tions. In cases where a large quantity of liquid inoculum could be pre- 

 pared, a very effective method of inoculation was to dip the whole top 

 of the test plant with its punctured leaves, keeping it submerged for 

 an hour or longer, with several shakings during the period. In a few 

 instances the test plants vv^ere so placed that the punctured leaves 

 remained buried in the soil of the pots for a day or two. Tests were 

 made of placing the plants under an air exhaust after soil water had 

 been applied to their leaf surfaces. The soil solution was centrifuged to 

 concentrate the canker organisms when they were very few% but this 

 was without definitely satisfactory results. Still another method ^ em- 

 ployed was to atomize the leaves with sterile water, sift over them the 

 rather 4ry soil to be tested, and keep the leaf surfaces moist for several 

 days by holding the plants in a moist chamber and by repeatedly atom- 

 izing them with sterile water. 



It was not apparent that any of the modifications of testing procedure 

 could be relied upon to give a larger percentage of infections than the 

 standard method, or to show the presence of P. citri when the standard 

 method failed to give positive results. 



1 This method was first used by Miss Clara H. Hasse, of this office. 



