132 BACTERIA IN RELATION TO PLANT DISEASES. 
On account of these undesirable results Hiltner was inclined to give up the use of such virulent 
cultures for practical agriculture and was deterred from doing so only by the idea that the better 
nourished plants in the open field would better withstand the bacteria than would potted plants. 
The results of field inoculations in 1902 justified this opinion. There might, however, be field con- 
ditions in which injury would result from very virulent cultures. 
The benefit to the plant does not take place by absorption of the bacteroids. In the case recorded 
where absorption occurred there was no observable increased growth of the lupins. 
It is desirable, therefore, to obtain bacteria with a degree of virulence sufficient to cause a strong 
root-infection yet not strong enough to cause injury. The best method of obtaining such cultures 
is as yet theoretical. Hiltner considers the solution of the problem to be possible by keeping the 
virulent bacteria on media as rich as possible in carbohydrates for some time before using them. 
It is evident at any rate that nitrogen assimilation and virulence do not always run parallel and 
that the solution of the problem here presented promises better results in the use of pure cultures. 
In 1902 a new method of seed inoculation was tried in which, by the addition of milk instead 
of water, the injurious action of the substance in the seed-coat was suppressed. The idea originated 
with Spiegel who obtained good results with clover in this way. 
Good results with lupin were obtained on the birch moor at Bremen not only with milk but 
with a 3 percent pepton solution and also with a solution containing 2 per cent grape-sugar and 3 
per cent pepton, to spread the bacteria on the seeds. When water was used in the direct inoculation 
of seeds complete failure resulted. The use of milk gave such good results with lupin that it seems 
desirable to experiment with it further. 
It remains to be proved how these means would act in other soils. ‘The evidence seems sufficient, 
however, to warrant the belief that the injurious action of the seed-coat substance may be overcome 
without a preliminary swelling or germination of the seeds. 
For the present it is recommended that for large seeded varieties the soaking of the seeds be 
avoided. Instead, they should be mixed with a corresponding amount of limed moor soil to which 
the bacteria have been added and which has been adapted to this use by the addition of grape-sugar 
and pepton. For small seeded sorts, such as clover and serradella, however, the methods of direct 
seed inoculation which have already proved successful are recommended. 
Dr. Moore’s bulletin on soil inoculation for legumes was published in 1905 after several years of 
experimental work. It opens with a summary of previous literature and closes with abstracts from 
reports of farmers favorable or otherwise. | From November 1902,-to November 1904, the U. S. 
Department of Agriculture sent out 12,500 packages of inoculating material. This inoculating mate- 
rial was dried on cotton. With the cotton were transmitted two small packages of nutrient substances 
with directions for use in preparing the cultures: One package contained sugar, magnesium sulphate, 
and potassium phosphate; the other ammonium phosphate. The contents of the first package was to 
be put into one gallon of clean water along with the cotton holding the dried bacteria, the ammonium 
phosphate was to be added 24 hours later and the fluid held till it became well clouded. 
The bacteria were plated out and grown on 1 per cent agar containing 1 per cent maltose, 0.1 
monopotassium phosphate, and 0.02 mag. sulphate in roo cc. distilled water, and are said to become 
much more virulent on this medium than on that containing peptone or other nitrogenous substances. 
Field experiments by the acre are said to have demonstrated the much greater nodule producing 
power of the organisms grown on the non-nitrogenous media. 
‘“‘As the result of numerous trials, however [which are not described], it has been found that 
although the bacteria increase most rapidly upon a medium rich in nitrogen, the resulting growth 
is usually of very much reduced virulence, and when put into the soil these organisms have lost the 
ability to break up into the minute forms necessary to penetrate the root hairs. They likewise lose 
the power of fixing atmospheric nitrogen.” 
The optimum temperature for growth is 23° to 25° C. The maximum 40° C. The minimum 
10° C., ‘‘below 10° C. practically no multiplication took place.” Airis necessary. Cultures in sealed 
tubes deteriorated rapidly. ‘The bacteria in the soil will stand any degree of acidity or alkalinity 
that is not prejudicial to the host-plants. Potassium and sodium salts tend to inhibit the formation 
of the bacteria. Calcium and magnesium salts greatly favor their production. 
“ Alkaline nitrates in the proportion of 1 to 10,000 are sufficient to prevent the formation of 
nodules. * * * the cultivation of the bacteria upon media containing appreciable quantities of 
nitrogen for any length of time is sufficient to cause them to lose both the power of infection and that 
of fixing atmospheric nitrogen.” 
Following Peirce the organism is regarded as a parasite, the doctrine of symbiosis being dis- 
credited. The host-plant is supposed to obtain its nitrogen by absorbing the bacteria. Ideas similar 
to Hiltner’s are expressed, e. g., that potassium nitrate in cultures reduces their power to produce 
nodules, that plants may bear inactive nodules and that ‘‘there is every reason to believe that when 
