TECHNIQUE FOR INOCULATING PINE SEEDLINGS 

 WITH CRONARTIUM FUSIFORME 



G. A. Snow and A. G. Kais 

 Southern Forest Experiment Station, U.S. Department of 

 Agriculture, Forest Service, Gulf port a Mississippi, U.S. A 



ABSTRACT 



An apparatus has been devised for inoculating pines with uni- 

 form numbers of Cronartium fusiforme basidiospores . Inoculum 

 densities are regulated by adjusting the time that seedlings 

 are exposed to an airstream which carries the spores and by- 

 regulating the rate of airflow. The method has produced 

 99 percent infection of pines. 



.An apparatus for applying uniform amounts of basidiospores to test 

 plants has been developed at our laboratories in Gulfport. It is currently 

 being used to inoculate pine seedlings with Cronartium fusiforme Hedge. $ 

 Hunt ex Cumm. It has been described before (Snow, 1968) , but several 

 modifications have improved its efficiency. 



The device consists of two plexiglass boxes connected by a brass 

 tube (Fig. 1) . After telia on oak leaves have been induced to sporulate 

 in the large box, individual pine seedlings are placed in the small box 

 with the tuft of juvenile needles against the connecting tube. A vacuum 

 line to the small box creates a flow of moist air that carries falling 

 spores to the pine seedlings. The air entering the system is moistened by 

 forcing it through gas-dispersion tubes in two bottles of water. A 

 bleeder valve in the second bottle improves uniformity of airflow. 



Before plants are inoculated, a glass cover slip is exposed to the 

 airstream (2 mm from the connecting tube in the small box) for a measured 

 time and at a given rate of airflow. The cover slip is examined to 

 determine the number of spores per square millimeter. Spore density is 

 then regulated by adjusting time of seedling exposure and rate of airflow. 

 Adjustment is accomplished with a timer for the vacuum pump, and an air- 

 flow regulating valve. Calibration runs and readjustments are made after 

 8 to 10 plants are inoculated in succession. Spore densities of 30 to 60 

 spores per square mm have been maintained in several experiments (see Kais 

 and Snow, a second paper in these proceedings) with exposures of 5 to 60 

 seconds per seedling at airflow rates of 3 to 10 liters per minute. During 

 inoculation the plexiglass boxes are held in a cabinet that is maintained 

 at 20+0.1 C and is continuously moistened with a humidifier. Seedlings 

 are also atomized with distilled water before and after inoculation. The 

 inoculated seedlings are transferred to moist chambers in the cabinet and 

 are incubated in these chambers for 24 to 48 hours. 



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