116 BACTERIA IN RELATION TO PLANT DISEASES. 
This they did by means of repeated inoculations on bean plants. The first infection produced inactive 
nodules. Bacteria from these nodules, kept during the winter on gelatin containing an extract of 
leguminous plants, when inoculated on bean plants produced comparatively active nodules, while 
‘the nodules formed by them on peas were less active then those due to normal pea-bacteria. The 
following table gives the results for two average plants in each experiment: 
4. Uninoculated’ bean plants: .< ..< cimis <5 016 ::0.4 sa.2'6 ora, certo dteein waar o nodules. 
2. Bean plants inoculated with normal pea-bacteria............ 255 
3. Bean plants inoculated with pea-bacteria from bean nodules... 446 
4. Bean plants inoculated with bean-bacteria................46- 575 
From this it is plain that the three cultures of bacteria possess very different degrees of 
virulence for the bean. 
Hiltner says that he is almost completely convinced that the various nodule bacteria are only 
adaptation forms, a conclusion which is likewise reached by Mazé, who recognized two groups, differ- 
ing in the reaction of the soil in which they are found, capable of infecting only plants native to soil 
of the same reaction, but capable also of being transformed by gradual alteration of their nutrient 
substratum. However, the failure to obtain cross-inoculations from beans to locusts, both native 
on calcareous soils, does not bear out Mazé’s statement that nodule bacteria from calcareous soils 
can infect all lime-loving plants. 
According to Hiltner, the amount of inoculating material used does not influence number, size, 
or activity of nodules. He used an emulsion of distilled water and bacteria from pure cultures, in 
one case 100 times stronger, in the other case 100 times weaker than his normal mixture. The plants 
tried were vetches. The results were equal, except that in the case of large doses the roots remained 
smaller, probably because the plant had to exert more energy to keep the root-nodule formation 
normal. 
When a given amount of inoculating material was applied (a) at once (b) in 3 doses, no constant 
difference in nodule formation was observed. If there were fewer nodules on a particular plant they 
were individually larger. In all cases the amount of nodule formation bore a definite and constant 
relation to the aerial parts of the plant. 
The varying degrees of virulence, and hence the differences in the effects produced by the same 
bacterium, were not formerly understood. Thus nitragin often failed to produce results because the 
bacteria used were from nodules produced by weakly virulent forms. At other times it was eminently 
successful because virulent bacteria chanced to cause infection in the nodules used in its preparation. 
To make nitragin really successful, cultures should be made from the nodules of plants grown in 
soil which has repeatedly borne the same species, and whose bacteria have, in consequence, lived 
repeatedly in the nodules of that species. 
Although the balance between plant and bacteria is not disturbed by the amount of inoculating 
material used, it is altered by the quality of such material. Active nodules make the plant immune 
against bacteria of the same or of lower virulence than the ones which formed the nodules. Only 
bacteria of higher virulence are able to penetrate the roots. This fact is said to stand alone in the 
plant world. 
Hiltner observed that Robinia plants like other Leguminosae, produced only small, inactive 
nodules when grown in nutrient solutions which covered the roots completely. When, however, 
some of the solution was poured off a strikingly rapid growth took place in the nodules on the parts 
of the roots above the solution, increasing the growth of the plant in a marked degree, while the 
submerged part remained free from nodules, even when repeatedly inoculated with pure cultures 
and with the contents of nodules. His two figures are very striking. 
This result, which supports the theory of immunity is also in agreement with the observed fact 
that, in the soil, nodules are not evenly distributed on the roots, but appear on the parts of the roots 
nearest to the surface of the ground, and decreasing in size and number as they go down. ‘This 
freedom from infection on the lower roots is due, Hiltner claims, to the immunity brought about by 
an early infection of the upper ones. When for any reason, nodules do not appear on the upper 
roots, they form later on the lower ones. A general distribution of the nodules results from inocu- 
lation after the growth of the plant is well advanced. 
The location of nodules is independent of their oxygen requirements. ‘The formation of nodules 
ceases only because a larger number would disturb the balance between bacteria and plant. It is not 
difficult to induce them to form on the deeper roots if those only are inoculated. 
It is useless to inoculate open fields with nodule bacteria, unless the soil is free from them or 
contains only forms of lessér virulence than the ones used for inoculation. It has been repeatedly 
demonstrated that soils free from nodule bacteria do exist. ‘Therefore Mazé’s claim that bacteria are 
present but lack the necessary developmental conditions seems unjustified. Equally unjustified are 
the opinions of those who leave out of consideration the question of virulence. 
