292 MANUAL OF MICROBIOLOGICAL METHODS 



is accomplished by placing a paper on a caged plant to be inoculated 

 and by laying on this paper a portion of a diseased leaf which carries 

 aphids. As the new leaf tissue dries, the insects crawl over the paper 

 to the fresh leaf below. When insects are involved, a variety of special 

 cages (Leach, 1940) may be necessary. 



All stages in the life cycle of the insect employed must be considered 

 because inoculation capabilities often vary in this respect. The insect 

 should be identified by a competent authority, and if significant results 

 are obtained a specimen should be deposited in a permanent reference 

 collection. 



A detailed discussion of methods for studying insect transmission 

 has appeared (Leach, 1940). Some knowledge of the mouth parts of 

 insects and of their feeding and breeding habits is necessary if insects 

 are to be used successfully for inoculating bacterial plant pathogens. 

 Sometimes they merely open infection courts for the microorganisms 

 to enter. Aphids and leafhoppers are particularly important as car- 

 riers of virus diseases. 



Before claims are made about insect transmission of a plant disease, 

 demonstrations of the following (Leach, 1940) seem a minimum for 

 proof: (1) close but not necessarily constant association of the insect 

 with diseased plants, (2) regular visits by the insect to healthy plants 

 under conditions suitable for the transmission of disease, (3) presence 

 of the pathogen or virus in or on the insect in nature or after visiting 

 a diseased plant, (4) experimental production of the disease by insect 

 visitation under controlled conditions and with adequate checks. 



Fungus inoculation. In general, inoculations with the spores or 

 mycelia of fungi differ only in detail from those made with bacteria. 

 For pathogenic fungi, variations in the mode of entrance and in other 

 important characters require modified procedures. Some of the more 

 common methods are discussed by Riker and Riker (1936). 



Virus inoculation. Brief mention is given to inoculations with viruses 

 without implication that they are microorganisms. Experimental 

 inoculations are more commonly accomplished by mechanical processes, 

 insects (see ''Insect Inoculation"), and grafting. 



Mechanical inoculation of a virus frequently is made by grinding 

 diseased tissue in a mortar with a little water and by rubbing the juice 

 lightly over leaves of the host plant. With some viruses, the following 

 modifications may be helpful. A favorable reaction between pH 7.0 

 and 8.5 may be obtained by placing a little 10 M K2HPO4 in the mor- 

 tar before the leaves are triturated. Sometimes viruses have to be pro- 

 tected from rapid oxidation by means of 0.5 per cent anhydrous Na2S03. 

 Just enough friction by a finger, cheesecloth, or similar agent is employed 

 \o injure the leaf hairs. With viruses diflftcult to transmit, better infeo- 



