278 BACTERIA IN RELATION TO PLANT DISEASES. 



In the roots the infection begins generally in the woody bundles of the central cylinder, and from 

 there spreads to the secondary wood; and the attacked elements (vessels, fiber, etc.) color as usual 

 either yellow or brown, becoming filled at the same time with the clotted, blackish substance. 



In tlic leaves the infection manifests itself by the pale spots above described, which are due to 

 the disorganization of the chloroplasts through work of the microorganism, which multiplies rapidly 

 in the cell. With the advance of the disease the protoplasm of the cell contracts and shrinks, caus- 

 ing the collapse of the cell walls; from this time on the tissue decays and there appear in the mesophyl 

 tin- brown and depressed spots which we have already noted on the petiole in advanced disease. 



Microscopic examination shows in the cells of the diseased tissues numerous motile microorgan- 

 II), isolated and united in colonies, but does not reveal any trace of mycelium. 



We have cultivated this organism in many nutritive media, proceeding in the following manner: 

 We took small pieces of the leaves, branches, stalks, and roots, carefully washed them with water 

 and soap, and afterwards disinfected them with a solution of 1 : 1000 mercuric chloride; they were 

 then put into sterile distilled water and passed finally through alcohol and ether. 



The pathological pieces thus treated were put into tubes containing nutritive media of our 

 preparation, and from all the infected organs thus treated and sown in the different nutritive media, 

 we obtained always the development of a particular microorganism, which presented the following 

 morphological and cultural characters: 



Microscopic aspect and color ability. This microorganism has the form of a small rodlet length 

 2 to 4/u, width 0.4 to o.6ju, with the ends slightly rounded. It stains well with all the anilin stains, 

 even cold, but especially with gentian violet; it resists Gram completely. 



In broth cultures we observed vivacious vibratory and rotary movements of the microorganism, 

 which appeared in the form of rodlets or of spherules, according to the position in which observed. 



Relation to oxygen. In anaerobic conditions the microorganism does not develop at all, or only 

 with great slowness, which indicates that it is prevailingly aerobic. 



Behavior in regard to temperature and to nutritive media. It develops well at room temperature 

 (15 C. circa), but more rapidly in the thermostat in the various nutrient media, and especially in 

 neutral conditions. 



Gelatin cultures. In 24 hours at room temperature it forms, in stab-cultures, a cup of initial 

 liquefaction which progresses rapidly in cylindrical form until all the gelatin is liquefied. On the 

 surface of the culture there is then formed a pellicle sufficiently thick that it detaches itself on agita- 

 tion, falling to the bottom as a whitish, mucilaginous precipitate, and the liquid becomes a beautiful 

 light green color in the upper part of the tube. 



Agar. On glycerinated streak-agar cultures there develops, after 48 hours of incubation, a 

 whitish surface growth little extended and slightly elevated, not shining. 



In stab-cultures the development is still more scanty; after 4S hours' incubation, the stab-growth 

 is scared v visible; growth soon ceases. 



In plain agar the organism develops rather better. In streaks, in 24 hours, it forms a wet- 

 shining, surface growth, rather elevated, with lobed margin, of a whitish color, and with a tendency 

 to occupy all the free surface of the tube. In stab-cultures the growth extends to the bottom of 

 the tube, at the surface enlarging into the form of a whitish disk, in the center of which appears in 

 time a roundish, yellow spot. 



Broth-cultures. In alkaline broth it develops poorly, with slight turbidity and formation of a 

 scanlv deposit; in the upper part of the tube the liquid assumes a pale green color. 



hi plain broth the development is more rapid and vigorous; in 48 hours of incubation the entire 

 culture is uniformly clouded, with an abundant dirty white sediment, which in time becomes a pale 

 yellow. At the surface and along the walls of the test-tube a pellicle forms which detaches itself 

 easily; the liquid assumes a light green color. 



Potato-cultures. At the temperature of the incubator there develops, in 48 hours, a gray-white 

 growth, wet, rather elevated, with a tendency to spread. With age the culture assumes a granular 

 aspect with a pale yellow color, tending toward brown. 



Chemical activity. In the tubes an offensive gas develops, but not hydrogen sulphide, as we 

 wire able to verify by exposing above the cultures a small strip of lead acetate paper previously 

 exposed to the vapor of ammonia. 



. 1 rtijn ia! reproduction of the disease. To prove that the disease was really due to the pathogenic 

 action of the microorganism of our isolation, we infected some vigorous plants of Matthiola, spraying 

 tin 111 with broth cultures, very much diluted. 



At the end of a few davs we obtained the reproduction of the disease on the aerial part of the 

 plants sprayed, with the same external characters and the same anatomical alterations (in the leaves 

 and in the young branches) which we have described in the plant naturally diseased. 



