December 22, 1923] 



NATURE 



90: 



Minute "Organisms" isolated from the Virus 

 of Mosaic Disease of Tomato. 



The nature of the infective principle in plants 

 suffering from mosaic disease is obscure, although 

 most recent workers favour the view that it is a living 

 organism. Allard and Duggar have emphasised the 

 minuteness of the causal agent, whilst Matz, Kunkel, 

 Nelson, and others have described protozoan - like 

 bodies in the cells of affected plants. 



A considerable amount of work has been done on 

 this problem at Cheshunt, and the present note records 

 the isolation and growth in pure culture of a minute 

 " organism " from the filtered virus of tomato mosaic. 



Isolations were made from affected plants by a 

 modification of Noguchi's method. Tubes of sterile 

 extract of tomato stem and leaf (loo gm. fresh material 

 to 1000 cc. distilled water) were prepared, and into 

 each was dropped a small piece of living tomato 

 tissue cut, under aseptic conditions, from the interior 

 of healthy green fruits. Ten tubes were inoculated 

 by touching the tomato tissue under the liquid with 

 a loopful of tomato virus filtered aseptically through 

 a sterile Doulton candle. Ten uninoculated tubes 

 \\ ere left as controls. All tubes were incubated in a 

 Bulloch's anaerobic jar for two months, and on re- 

 moval were found to be clear. The tubes were then 

 left under ordinary atmospheric conditions, and two 

 months later one tube was contaminated by a fungal 

 growth, but the liquid in the remaining nineteen was 

 quite clear in both the inoculated and the control tubes. 

 This liquid was examined for micro-organisms by 

 plating and streaking upon different culture media, 

 but no growth was observed. On the glass, however, 

 of each of the original inoculated tubes about one 

 centimetre above the liquid were small brown bodies, 

 the largest of which was 200^ in diameter. No such 

 bodies were present in the controls. 



These bodies were tightly fixed to the glass, and not 

 easily detached. They are brittle and break into 

 fragments of a crystalline appearance. The bodies 

 are discoid convex, and when stained with borax 

 armine the surface shows concentric and radiating 

 markings. They clear in acid with evolution of gas, 

 the cleared bodies having a fine granular appearance. 

 When stained by Giemsa's method they resemble bac- 

 terial colonies, containing deeply stained purple gran- 

 ules standing out distinctly on a stained background. 

 These granules are 0-3 to 0-4/^ in diameter (occasionally 

 smaller granules are seen) and appear as cocci, diplo- 

 cocci, polar bodies, or unstained rods. These are not 

 merely crystals or detritus but definitely organised 

 bodies growing in colony formation. Distinctive pre- 

 parations have been made by staining with Giemsa 

 for 24 hours and then differentiating with abso- 

 lute alcohol. The granules are best seen in smears 

 made from the cleared colonies prior to fixation by 

 drying. 



Tubes of virus kept in the laboratory for six to 

 ' ighteen months under aerobic conditions revealed 

 similar colonies on the glass in those tubes where no 

 toluene had been added for preservative purposes or 

 from which the toluene had disappeared. After acid 

 clearing and staining, the minute granules were readily 

 demonstrated. Films made from the clear liquid in 

 the tubes bearing the colonies were also stained with 

 Giemsa, and purple-stained granules similar to those 

 M) abundant in the colonies were regularly found in 

 I hesc preparations. They were not numerous, five or 

 :six only being seen in a single field, and appearing as 

 cocci or as diplococci. 



Continued cultivation of the " organism " has been 

 maintained in tubes of tomato extract containing 



NO. 2825, VOL. I 12] 



cubes of sterile raw tissue. (The addition of 0-3 gm. 

 calcium carbonate to 10 cc. of extract hastens the 

 production of colonies. Increased concentration of 

 carbon dioxide in the atmosphere also seems to assist 

 in the formation of colonies and alters their appear- 

 ance ; the brown colonies becoming white and chalky.) 

 Numerous media have been inoculated with negative 

 results, but one inoculation is especially interesting. 

 A flask of lemco gelatine containing a high proportion 

 of gelatine was inoculated with a drop from one of 

 the original culture tubes. No growth was apparent 

 for four months, but after six months the surface was 

 covered with minute hard white bodies, which on 

 examination proved to be similar to those described. 

 Colonies transferred to Noguchi tubes dissolved in the 

 liquid, and films prepared from this ten days later 

 showed the presence of minute granules either singly 

 as diplococci or as aggregates in alveolar plasmodium- 

 like structures in which cocci stood out deeply stained 

 in comparison with the faintly stained matrix. 



The bodies forming on the glass of Noguchi tubes 

 and in the liquid, and the lemco colonies, have been 

 inoculated into healthy plants under various con- 

 ditions ; and while there are indications that they may 

 be causally related to mosaic disease, no definite claim 

 can yet be made. The presence of these " organ- 

 isms " in the virus of tomato plants suffering from 

 mosaic, and their very interesting nature, appear, 

 however, of sufficient importance to warrant the im- 

 mediate direction of the attention of workers on this 

 difficult problem to their existence. A detailed in- 

 vestigation of the character and genetic relationships 

 of the " organisms " recorded in this note and their 

 relation to mosaic disease is being carried out at 

 Cheshunt. W. F. Bewley. 



Experimental and Research Station, 

 Cheshunt, Herts, December 3. 



Globular Lightning. 



Your correspondent, Mr. E. Kilburn Scott, suggests 

 in Nature of November 24, p. 760, that " the ball 

 may be a mass of concentrated nitrogen oxides," and 

 considers that this would " fit in well with the forma- 

 tion and action of such gases," and he compares the 

 chemical activity of lightning with the well-known 

 reactions occurring in high-tension arc flames. 



Although I do not wish to be understood as express- 

 ing any opinion regarding " globular " lightning, I 

 should like to point out that in the letter which 

 appeared in Nature of September 15, p. 396, I pro- 

 duced evidence in connexion with the extremely vivid 

 and prolonged thunderstorm of July 10, 1923, which 

 left no doubt that the chemical changes that occurred 

 then resembled those of the silent electric discharge, 

 rather than high-tension arc flames, because, although 

 there was no increase in the proportion of the oxides 

 of nitrogen in the air within the area of the storm, 

 there was a very great increase in the proportion of 

 ozone. 



I may add that since the proportion of nitrogen per- 

 oxide is always much higher in London than in country 

 air, and is considerably greater in winter than in 

 summer, we may look, as in the case of sulphur di- 

 oxide, to combustion oif coal as the probable source of 

 most of it at least. The seasonal changes of the 

 curves for these two variable ingredients of the atmo- 

 sphere are very similar, and are not in any way related 

 to that for ozone. William C. Reynolds. 



*' Wharfedale," Upminster, Essex, 

 November 26. 



