426 STATE BOARD OF AGRICULTURE 



gation was to determine whether or not any organisms could be demonstrated 

 in the sieve tissues of mosaic plants. As the bean work progressed the scope 

 of the investigations was widened to include tomato mosaic, clover mosaic 

 and potato leaf-roll. 



Materials 



Bean mosaic material has been available in great quantities from a mosaic 

 seed plot which has been grown each year as part of the field experimental 

 work. This has included most of the common varieties of field and garden 

 beans, as well as Lima, yard long, adsuki, rice, urd, tepary, moth and Windsor 

 beans. All of these species showed distinct mosaic symptoms during the 

 summer of 1922, which was a very favorable season for the development and 

 spread of the disease. For a study of healthy tissues, seed from healthy 

 plants was grown under cheese cloth cages in the greenhouse. 



Tomato mosaic was common late in the summer and material was collected 

 from several gardens where practically all of the plants were diseased. Po- 

 tato leaf-roll was not found in great quantity but sufficient typical material 

 was secured for the purpose of this investigation. Clover mosaic was plen- 

 tiful and was used as a check upon the results obtained in studying bean 

 mosaic, since it has been shown that the mosaic disease of each can be recip- 

 rocally transferred. 



Methods 



In all of the early work on bean mosaic the ordinary botanical histological 

 methods were followed. Paraffin sections were stained with the stains most 

 used by botanists. Many thousand transverse sections were cut of leaf, 

 stem and petiole material imbedded in paraffin and studied under the high 

 powers of the microscope, but, aside from slight deviations, diseased plants 

 revealed nothing more than healthy ones. It was then decided to try other 

 killing and fixing agents and employ new stains. Various solutions used by 

 protozoologists were modified to adapt them to plant material, and protozoan 

 stains were used for coloring the sections. Small bits of stems, leaves and 

 petioles were taken from near the growing points of the plant and fixed for 

 12-24 hours in the killing and fixing solutions. The material was then washed 

 for 24 hours in running water, dehydrated and imbedded in paraffin in the 

 usual way. A radical departure in technique, and one which it is believed 

 would have illuminated the mosaic problem long ago if employed, was intro- 

 duced at this point. Longitudinal sections 10 microns thick were cut from 

 the paraffin material. From the photomicrographic illustrations in works 

 published on mosaic and related diseases it appears that the basis of these 

 studies has been mainly the study of transverse sections. Apparently little 

 work has been done in which longitudinal sections have been stained and 

 critically examined. There are probably various reasons for this. It is 

 very difficult to cut longitudinal sections of stem and petiole material due to 

 the lignification of the xylem elements and the presence of fibrous tissue in the 

 phloem. Much patience and a perfect condition of the sectioning razor are 

 essential to success in securing sections that are not badly mutilated. Since 

 the general assumption has been that the organisms were very small and gen- 

 erally distributed throughout diseased plants, the conclusion that transverse 

 sections should contain them was sound from this standpoint. However, the 

 results of the work presented here show why the cross section method has 

 failed. Transverse sections have proved, in the author's work, to be without 

 value so far as showing the location, position or nature of the organisms. 



