HISTOPATHOLOGICAL STUDIES 



While sonic work has been done on the development of Rhabdo- 

 cline pseudotsugae within infected needles (63), little reference is 

 made to the early stages of the fungus at, and immediately following, 

 the time of infection. Adequate reports of the incipient stages of 

 apothecial formation have not been published nor is detailed 

 description of the development of the hymenium available. The 

 purpose of this phase of the study was to investigate the histo- 

 pathologv of the Rhabdocline needle cast disease to provide the 

 answers to some of these problems. 



Diseased needles were collected, cut into short sections, and 

 immediately killed and fixed in formalin-acetic acid-alcohol (24). 

 Immediately upon reaching the laboratory, air was evacuated from 

 the tissue with a water aspirator. The material was stored in forma- 

 lin-acetic acid-alcohol for a minimum of 48 hours and then dehy- 

 drated in a tertiary butyl-ethyl alcohol series (24). A mixture of 

 equal parts of tertiary butyl alcohol and paraffin oil was used to 

 effect a gradual transfer from the alcohol to paraffin. Fisher Tissue- 

 mat (M.P. 56-58.5° C.) was used for both infiltration and final 

 embedding. After softening in a mixture of equal parts of glacial 

 acetic acid and 60 per cent alcohol for 36 hours, serial sections were 

 cut, 10 [i thick, and mounted on slides with Sass adhesive III (51). 



A stain combination of 1 per cent chlorazol black E in 70 per 

 cent ethyl alcohol and 1 per cent water-soluble solutions of safranine 

 O and crystal violet was used for all staining. The first two stains 

 delineated the needle structures, whereas the crystal violet differen- 

 tiated the fungus from the host tissue. 



NORMAL NEEDLE ANATOMY 



Douglas-fir needles are differentiated into dermal, mesophyll 

 and vascular tissues. A relatively thick protective cutin covers the 

 outermost or epidermal layer of cells. The remaining one to three 

 layers of cells in this region consist of thick -walled hypodermal cells. 

 These long, fiber-like cells are lignified and circumscribe the needle, 

 with the exception of the area containing the bands of stomata on 

 the lower surface. 



The mesophyll consists of large, irregular parenchyma cells 



45 



