362 R. T. BINGHAM 



PROBLEMS OF LARGE-SCALE INOCULATION 



CONTROLLING TEMPERATURE AND HUMIDITY 



To inoculate more than a few thousand seedlings the researcher usually 

 must use artificial inoculation in outdoor nurseries where young seedlings 

 are compacted in nursery beds or portable flats. For convenience and 

 accessibility, and to reduce time and costs of all progeny test treatments 

 and examinations, test nurseries are usually located near urban research 

 installations. Here the environment is likely to be warmer and drier 

 than that found in the forests. 



Due primarily to weather, often there is only a short period of time 

 available in the field for securing the best inoculum; furthermore, 

 naturally infected Rites spp. leaves are commonly the only adequate 

 sources of inexpensive inoculum. Also, the need to secure either a heavy 

 or at least uniform exposure of test plants requires that the investigator 

 inoculate all test seedlings simultaneously with the same batch of 

 inoculum. 



All of the above conditions apply to our inoculation situation at 

 Moscow, Idaho. In all of our white pine blister rust resistance progeny 

 testing we use white pine seedling progenies in a small and highly 

 specialized nursery. Knowing that this nursery was relatively warm and 

 dry, we delayed our first large-scale inoculations until the nursery 

 environment had cooled. Then, because of frosts, we were often too late 

 to collect the preferred inoculum--solidly-infected Ribes hudsonianum 

 Richards var. petiolare (Dougl.) Janz . leaves from this large-leaved, 

 streambottom plant. Instead, the less heavily infected leaves of 

 upland Ribes spp. (principally R. visoosissimum Pursh.) were used; these 

 upper-slope plants were protected from frost by the warm air of a hill- 

 side or a ridge-top temperature inversion zone (see Mielke, Childs and 

 Lachmund, 1937, for relative susceptibility of these Ribes spp.). 



Fortunately, a really hot early October inoculation season finally 

 occurred. In the second and third days of the 72-hour inoculation period, 

 temperatures in the inner inoculation chambers reached 85° F , and relative 

 humidity dropped below 95%. Thus, temperatures exceeded the 50-65°F 

 range reported as optimal in the eastern and central U.S.A. for C. ribioola 

 teliospore germination, for production and germination of basidiospores, 

 and for penetration of pine needles by the germ tubes thereof (Hirt, 1935; 

 Van Arsdel, 1954). In fact, the supposed maxima (approximately 70°F , 

 Hirt, 1935) for teliospore and basidiospore germination were exceeded. 

 On two occasions the relative humidity dropped below the supposed critical 

 level of 97% (Hirt, 1935); but despite this, standing water was maintained 

 on some pines and Ribes spp. leaves for at least two 12- to 16-hour periods 

 of presumably near optimal conditions (50-60°F , 100% R.H.) that occurred 

 overnight. Even with these conditions, after 2 years control seedlings 

 were 85% infected. 



As a result of these experiences we commenced inoculating earlier in 

 the season, i.e., from September 10 to 20 when higher quality R. hudsonianum 

 inoculum was available. In the last several years we have inoculated 

 during seasons when outside, mid-day temperatures exceeded 95°F and inner 

 chamber temperatures 87°F , with relative humidities below 85%. Two years 

 after inoculation we have found control plants 75 to 100% infected. Thus, 

 we can make highly successful, simultaneous inoculations of large progeny 

 tests given: (1) fairly spacious chambers (approximately 8 to 10 cu ft 



