80 BACTERIA IN RBIvATION TO PI^ANT DISEASES. 



Since the relation of the middle lamella to the dissolving enzym is of the greatest importance 

 to bacterial infection, tissues from different sources and from plants of different ages were tested. 

 Thin sections were immersed in an enzym solution from decayed potatoes and carrots. The plants 

 tested were potatoes, carrots, onions, roots of yellow turnips, red beets, apples, seeds of Phaseolus 

 vulgaris green and ripe, the pods of Phaseolus vulgaris both green and dried, date seeds, stem of 

 Phaseolus vulgaris, Viciafaba, Cucumis sativus, Zea mays, Avenea saliva, Brassica acephala, young and 

 old plants, stem and leaf stalks of Sblanum tuberosum, and Lycopersicum esculentum, leaf stalks of 

 Daucus carota, Petroselinum sativum and Apium graveolens. 



The results were as follows : At the end of several days ' immersion in the solution the stems of 

 maize and oats, and the endosperm of date were entirely unchanged. The breaking up of the tissue 

 of red beet was extraordinarily slow. At the end of 60 hours the first evidence of softening was 

 observed in the thin sections and it must for the present be considered a question whether a complete 

 breaking up of the tissue can occur. Of all the other plants tested, the parenchyma tissue was 

 completely broken up into single cells with unchanged cell-membrane, while on |the other hand 

 all the suberized and woody tissues were unaffected by an immersion of longdxuration in the enzym 

 solution. The youngest tissue was broken up most rapidly, thus sections of the green seeds of Phase- 

 olus vulgaris were disintegrated in about 20 minutes; those of the ripe ones in about 2 hours. Green 

 pods of the same plant were broken up much more quickly than those completely dried ones gathered 

 in the late fall. I^ikewise the parenchyma of stems of Brassica acephala was disintegrated more 

 easily in June than at the end of November. 



Experiments were then carried on to test the action of acidified juice from decayed plants 

 sterilized with formalin, on the tissues of a single plant, i. e., the carrot root. Acidification was 

 made with malic, tartaric, and hydrochloric acid. The juice was mixed with the acid, left standing 

 4 hours at room temperature, 18 to 20°, and then tested as to its dissolving power. Potato juice 

 was always used. This titrated + 5 to phenolphthalein. 



The action of the enzym is gradually diminished by the acidity of a solution titrating+io, 

 and it entirely ceases when the solution titrates over +20. Citric acid and hydrochloric acid have the 

 same effect whUe malic acid is seemingly not as effective. 



The enzym, even in a very weak dilution remains active. The rapidity of a solution of the 

 tissue decreases proportionally to the amount of dilution of the enzym solution. 



The power of the enzym to destroy the middle lamella is destroyed by boiling. A much lower 

 temperature is also sufficient for this. Experiments show that the action of the enzym is destroyed 

 if kept at 60° for any length of time. 



According to the investigations of various experimenters the middle lamella is composed of the cal- 

 cium salt of pectic acid. Experiments were carried on to show whether this could serve as a nutrient 

 substance for the bacteria. Calcium pectate from carrots and yellow beets to which was added vari- 

 ous nitrogenous substances was inoculated with the bacteria. A nutrient solution containing 2 grams 

 of KH2PO4 and I gram of MgS04 to the liter was used. As sources of nitrogen i per cent asparagin, 

 peptone, potassium nitrate and ammonium phosphate were added. If the medium was not neutral 

 it was neutrahzed with soda, — temperature 28°. The flask containing the saltpetre showed no 

 growth. On the contrary, in all the others there was very evident growth in 48 hours. The gela- 

 tinous content of the flask cleared in the upper portion to a thin liquid, which was sharply differen- 

 tiated from the ever diminishing pectate layer. In 14 days the flask containing ammonium phosphate 

 was slightly cloudy throughout and the contents were changed into a thin fluid with a small amount 

 of precipitate. The fluid had a slightly aromatic odor, was weakly alkaline and very quickly dissolved 

 into their elements sections of carrot treated with 0.2 per cent formalin. The solution of the calcium 

 pectates was somewhat slower in the flask containing the asparagin, and stUl slower in that containing 

 the peptone. The contents of the control flasks remained gelatinous. The fluid in the flask con- 

 taining the ammonium salt gave the following reactions: 



Absolute alcohol No precipitation. 



Dilute hydrochloric acid No precipitation. 



Alcohol + hydrochloric acid No precipitation. 



Boiled with Fehling's solution No reduction. 



Boiled with concentrated hydrochloric acid Furfurol reaction. 



Boiled with concentrated hydrochloric acid and with Fehling's solution Very strong reduction. 



Slightly heated with dilute caustic potash lye Yellow color. 



On evaporating, a brown horny mass remains. The solution then contains a body, one of the 

 pentoses — perhaps one of the hexose group — which is distinguished from calcium pectate only by 

 its solubility. It is thus probable that the calcium pectate has been converted into metapectate 

 by the enzym of the bacterium. A further splitting up of the metapectate into sugar does not appear 

 to take place outside of the bacterial cell. Alcohol is also present, apparently. At least the solution 



