THE CUCURBIT WILT: CULTURAL CHARACTERS 139 



you determine the kind of acid produced from cane-sugar? 

 Use large flask cultures containing peptone water cane-sugar and 

 calcium carbonate. Be certain that they contain only this one 

 organism i.e., plate out just before undertaking isolation of the 

 acid, and test on young cucumber plants. Study the viscidity 

 in cultures : compare it with that of Bacterium leguminosarum. 



Non-nutritional Environment. — Effect of heat, of sunlight, 

 of dry air (killed quickly), of chloroform in bouillon, of weak 

 acids, of salted bouillons? Can you get any growth in bouillon 

 at 9°C., or at 37°C.? How many months will the organism 

 live dry on cucumber seeds? 



For the disease. Signs. — Contrast cucumbers and 

 squashes. Observe period of incubation; time between local 

 appearance of disease on inoculated leaf and general infection 

 of the plant. What are the most conspicuous external in- 

 dications of this disease? Can you produce it on watermelons? 

 on gourds? Write a description of it. 



Histology. — How many centimeters in advance of external 

 signs on the inoculated leaf can the bacteria be traced down the 

 petiole? Begin before the wilt has attacked the whole leaf- 

 blade. With a hot knife sever the petioles where they join 

 the stem. Twenty or more plants will be necessary for this 

 experiment, which is best performed as soon as a few square 

 centimeters of the leaf show the characteristic wilt. After re- 

 moval of the inoculated leaf, what per cent of the plants remain 

 free from the disease? In tracing the organism from the leaf- 

 blade into the stem, determine what tissues are occupied. Are 

 cavities formed? Is cellulose destroyed? Is the wilt due to 

 lack of water-supply or to toxic action? Are the bacteria in 

 the leaf-blade confined to the vessels or do they invade the 

 leaf -parenchyma? Do you think they swim through the vessels 

 or only grow through them? What are your reasons? 



Make cross- and longitudinal sections of the infected stems. 

 If time permits, fix, stain and mount. Which vessels of the 

 stem are first occupied? Why? What tissues of the stem other 

 than vessels are occupied and destroyed? 



Can you demonstrate the organism in the phloem? In 

 the interfascicular parenchyma? Consult Figs. 72 to 78. 



