BACTERIAL WILT OF CUCURBITS. 23 



DISSECTION EXPERIMENTS. 



It is clear from the foregoing that striped beetles in the active state 

 are sometimes capable of harboring the wilt organism for consid- 

 erable periods of time. With the idea of determining in what part 

 of the body the bacteria are carried, the following beetle dissection 

 experiments were carried out. 



Six 12-spotted cucumber beetles were fed for three weeks on wilted cucumber 

 plants in pots and finally for five days on cut stems and leaves of wilted plants. After 

 this last feeding (Aug. 8, 1917) the heads and legs of the beetles were removed sep- 

 arately with sterile instruments. Then the anal region was cut off with a hot scalpel, 

 and the intestinal contents were carefully squeezed out with sterile instruments on 

 a sterile Petri dish, using care that none of the contents should come in contact 

 with the exterior of the insect body. Young potted cucumber plants were then 

 inoculated separately with heads and mouth parts, legs, intestinal contents, and repro- 

 ductive systems. In each case the body parts named were pricked into the young 

 leaves without moistening the surfaces. Five days later one leaf inoculated with 

 intestinal contents had started to wilt. After two days more, wilt had started in one 

 leaf inoculated with beetle legs and in two additional leaves inoculated with intestinal 

 contents. This infection from beetle legs is not to be wondered at, as the beetles were 

 allowed to feed on cut stems and leaves of wilted plants for several days prior to dis- 

 section. These plants had been kept throughout the test in a large 6-foot square 

 beetle-free cage (PI. II, fig. 1) and were entirely free from insect gnawings. A hundred 

 or more uninoculated cucumber seedlings of the same age and variety held as controls 

 in the same cage remained free from the disease. 



On August 16, 1917, the preceding experiment was duplicated, using six striped 

 and twelve 12-spotted cucumber beetles. Five days later one of the plants inoculated 

 with intestines and one inoculated with legs of the 12-spotted species were found with 

 wilt. In this case none of the plants inoculated with head parts became infected. 



During the summer of 1918 four more sets of dissection tests were carried out. On 

 June 15, after a 7-day's feeding on wilted plants, forty striped and three 12-spotted 

 cucumber beetles were dissected as in preceding experiments, and separate cucum- 

 ber seedlings were inoculated with heads and with intestinal contents. Ten of the 

 forty striped beetles were feeding on wilted leaves when taken. The other thirty were 

 not feeding when taken for dissection. In these cases the body parts were crushed in a 

 drop of tap water and pricked into the cucumber leaves. In 11 days two plants and 

 next day three more plants showed wilt starting from leaves inoculated with intestinal 

 contents. Of these five wilted plants only one was infected from one of the ten beetles 

 feeding on wilt when taken; the other four wilted plants were from four of the thirty 

 beetles not feeding when taken. Cultures made from one of these plants gave Bacillus 

 tracheiphilus, and all five showed typical wilt symptoms. No infections resulted from 

 the three 12-spotted beetles. 



This experiment was repeated on June 28, using 45 striped beetles. In eight days 

 two plants were wilting from beetle-intestine inoculations and the following day two 

 more from intestines and one from head and mouth parts. Cultures were made from 

 the first two plants showing wilt, and Bacillus tracheiphilus was isolated and proved 

 to be virulent by successful inoculations. 



Sterile instruments were used and great care was taken in all these tests not to allow 

 the intestines to come in contact with external body parts. However, in order com- 

 pletely to eliminate all possible source of external contamination, the beetles in the 

 following two tests were immersed for five minutes previous to dissection in a 1 to 1 ,000 

 solution of mercuric chlorid in 25 per cent alcohol. The alcohol was added to facili- 



