-10- 

 inoculation of peas (Pisum sativum 'Alaska', 'Ranger', and 

 'Little Marvel') and N^ benthamiana . 



Freshly sectioned orchid tissues were exposed to antisera 

 (refer to chapter 3) conjugated with tetramethyl rhodamine 

 isothiocyanate (TRITC), and were examined for virus-induced 

 inclusions with a Nikon Fluophot fluorescence microscope 

 (Hiebert et al . , 1984). 



Results 

 Odontoglossum Ringspot Virus 



Crystalline stacked plate and paracrystalline inclusions 

 typical of ORSV and some other tobamoviruses (Christie and 

 Edwardson, 1977; Edwardson and Zettler, in press) were readily 

 observed by light microscopy in epidermal and mesophyll tissues 

 of infected plants (Figs. 2 and 3). These inclusions are 

 aggregations of virus particles (Figs. 4 and 5), as determined 

 by electron microscopy of thin sections. 



Thick -walled tissues required prestaining at room 

 temperature for 10 minutes followed by heating at 60 C for 5 

 minutes to achieve stain penetration. Treatment with the O/G 

 combination without heat resulted in poor staining of 

 inclusions (Fig. 6). Tissues were stained with Azure A for 15- 

 20 minutes at 60 C to detect the nucleic acid in inclusions. 

 These inclusions were stained reddish-violet (Figs. 8 and 9), 

 but they did not stain with Azure A at room temperature (Fig. 

 7). Heat treatment (60 C for 1-2 minutes) has also been shown 

 to be necessary for staining crystalline, paracrystalline, 

 angled-layered-aggregate and fibrous mass inclusions of other 



