INOCULATIONS WITH BACTERIA CAUSING PLANT DISEASE 291 



are difficult to wet because of a waxy covering, the surface can be gently 

 rubbed with a moist cloth. For work on a large scale, the suspension 

 of the organism can be made in a solution of a spreader (e.g., castile 

 soap, 1:1,000) to reduce surface tension. The concentration varies 

 according to requirements. Some spreaders, however, are toxic. (3) 

 A reduced oxygen supply may be important if the pathogen is a faculta- 

 tive anaerobe. For example, the protective wound-cork formation 

 in potato tubers requires abundant oxygen, while certain bacterial 

 pathogens, such as Erwinia carotovora (Jones) Holland, grow well with 

 little oxygen. (4) Water pressure, suction, prolonged spraying, and 

 other means can be used to saturate the intercellular spaces below the 

 stomata and thus to improve the penetration of bacterial suspensions 

 into these regions. This is particularly important with a pathogen, 

 like that causing black fire of tobacco (Johnson, 1937), which is often 

 not aggressive. With this method it is possible to induce necrotic areas 

 on plants not ordinarily considered hosts of the microorganism used. 

 Since bacteria that are usually considered saprophytes have caused 

 damage under these circumstances, care is necessary while interpreting 

 such results. For example, such saprophytes would hardly fulfill the 

 first of Koch's postulates, as given earlier. 



Wound inoculation. Suspensions of bacteria and small portions of 

 culture or of diseased tissue can be introduced into healthy plants through 

 wounds when they do not readily gain entrance through natural openings 

 or when heavier or more rapid infection is desired. The simplest pro- 

 cedure is to smear the point of a dissecting needle with the bacterial mass 

 and to insert the needle into the plant tissue. If large numbers of inocula- 

 tions are to be made, various instruments are useful. For example, 

 Ivanoff (1934) has described an inoculator in detail. It consists of a 

 hypodermic needle (size varied according to needs) with end closed and 

 smooth-walled opening made above the point, a suitable chamber to 

 hold a bacterial suspension, and a valve to regulate the flow. This 

 needle with a side opening may be used with an ordinary syringe. The 

 common hypodermic needle when pushed into a plant clogs too easily 

 to be practical. 



Known small numbers of bacteria may be introduced into micro- 

 wounds by means of a micromanipulator. Such wounds may resemble 

 those made by insects (Hildebrand, 1942). 



Insect inoculation. The translocation of microorganisms causing 

 plant disease and their introduction into susceptible plants by insects are 

 large and relatively undeveloped fields. The simplest technique with 

 active insects like cucumber beetles or leafhoppers is merely to place the 

 plant to be inoculated in the same insect cage with an infested diseased 

 plant (Leach, 1940). 



For virus diseases, inoculation with slow-moving insects, like aphids, 



