INOCULATION KITH SOUTHERN FUSIFORM RUST 329 



The humidified air is then mixed with the cool air in three 2.8-liter 

 Fernbach flasks which also serve to accumulate excess moisture. Tempera- 

 ture control is accomplished by balancing airflow from the humidification 

 and the cooling flasks without altering the total airflow (280 LPM) through 

 the system. The leads from the humidification flasks and those from the 

 Fernbach flasks to the inoculation chamber are connected so that the 

 chamber is not directly influenced by any one airstream. A scanning tele- 

 thermometer, chart recorder, and 11 temperature probes located at various 

 points in the inoculation chamber are used to determine that the tempera- 

 ture is held at 21+0.5 C throughout the chamber when set up in an air- 

 conditioned laboratory. Further temperature control is easily accomplished 

 by placing the system in a controlled environment chamber. 



The temperature-controlled, saturated air from the Fernbach flasks 

 is passed into the upper portion of the inoculation chamber through 0.63 

 cm Nalgene "T" connectors. A row of six, small (1 mm), evenly placed 

 holes was made with a hot needle on the facial surface of each "T" and 

 both ends were plugged with silicone rubber. 



Twelve internal ports (10 cm long by 5 cm wide by 6.25 cm high) made 

 of "Plexiglas", six equally spaced on each longitudinal side of the inocu- 

 lation chamber, contain telia-bearing leaves. Each port contains two 

 modified Nalgene T's. The "Plexiglas" port covers are large enough (5 cm 

 x 10 cm) to hold 24 0.85 sq cm discs of telia-bearing leaves. The leaf 

 discs are preconditioned for 5 or 6 hours at 20 C in a saturated atmos- 

 phere and then attached with 2% (w/v) water agar to the port covers so 

 that the telia are suspended directly over the incoming airstream. 



The inoculation chamber is made of 0.63 cm "Plexiglas"--1 . 27 m long 

 by 63.5 cm wide by 30.6 cm high. The chamber is large enough to hold 

 12 plastic plant-flats (18 cm x 23 cm x 5 cm) containing 6- to 8-week- 

 old pine seedlings. Each flat can hold from 20 to 80 seedlings, depending 

 on whether or not the seedlings are to be transplanted after inoculation. 

 With this system it is possible simultaneously to expose from 240 to 960 

 pine seedlings to the same sporidia density. After inoculation, the 

 seedlings are immediately transferred to an incubation chamber with a 

 saturated atmosphere at 20 C for 18 to 24 hours. 



A Kramer-Collins spore sampler (Kramer and Pady, 1966) placed in the 

 center of the chamber determines the number of sporidia per hr/ft 3 of air 

 sampled. Two-week-old telia of C. fusiforme , approximately 700 telia per 

 port, and an inoculation period of 8 hours provide a total inoculum 

 density of approximately 20,000 sporidia/ ft 3 . By adjusting the number of 

 telia per port, densities as high as 50,000 to 100,000 sporidia/ft 3 can 

 be attained. These densities are well in excess of those recorded under 

 natural conditions (Snow, 1968a, c). 



Although evaluation of this system is still in progress, completed 

 tests indicate that inoculum density can be closely controlled by con- 

 trolling the telial density on oak leaves, telial age, and number of 

 telia per port. The relation of inoculum density to infection is now 

 being studied to determine the optimum density for future studies utilizing 

 this system. 



