Dec. 12, 1889] 



NATURE 



141 



in the Forestry School, Royal Indian Engineering College, 

 Cooper's Ilill. 



In the Philosophical Transactions for 1887 (vol. clxxviii. B, 

 pp. 539-562, Pis. 32 and 33) the author published the reirults of 

 some investigations into the structure and nature of the tuber- 

 cular swellings on the roots of Vicia Faba and other Leguminous 

 plants. 



The chief facts established in that paper were as follows : — 

 That the tubercles occur in all places and at all times on the roots 

 of Papilionaceous plants growing in the open land, but that in 

 sterilized media and in properly conducted water-cultures they 

 are not developed, unless the root is previously infected by 

 contact with the contents of other tubercles. In other word=, 

 the tubercles can be produced at will by artificial infection. The 

 author also showed that the act of infection is a perfectly 

 definite one, and is due to the entrance into the root-hair of a 

 hyphalike infecting tube or filament, which starts from a mere 

 brilliant dot at the side or apex of the root-hair, passes down the 

 cavity of the latter, traverses the cortex of the root from cell to 

 cell, until its tip reaches the innermost cells of the cortex, where 

 it branches and stimulates these cells to divide and form the 

 young tubercle. 



These facts of the infection were entirely new, as were the 

 methods, and the author showed actual preparations of the 

 infecting filaments parsing down the root-hairs, at the time 

 (June 1887). 



In this paper the author described and explained the trumpet - 

 shaped enlargements of the filaments, and the bacterium-like 

 contents of the cells (bacteroids — gemmules^, and showed that the 

 latter arise from the former. He also pointed out that the root- 

 hairs are distorted at the point of infection, and that the infect- 

 ing filament originates there from a brilliant granule, presumably 

 one of the bacteroids. Another important observation was that 

 the protoplasm of the cells of the tubercle is stimulated by the 

 activity of the bacteroids in it, and behaves like a plasmodium. 



The author now draws attention to some results of his further 

 researches into this confessedly difficult subject. 



After numerous culture experiments and observations made 

 last year (1888), it was decided to abandon the broad-bean as 

 the subject for histological analysis, chiefly because it takes so 

 long to exhaust its stores of reserve materials ; it was better for 

 the cultures to be made with the pea, the cotyledons of which are 

 so much smaller, and the plant of which is more easily managed 

 in every way in water and pot cultures, while the tubercles and 

 their contents present no essential features of difference. 



But more conclusive evidence than the above is offered for the 

 identity of the bacteroids in the two cases. In some of ihe cultures 

 made in the summer of 1888 the roots of the pea were success- 

 fully infected with bacteroids taken from the tubercles of the 

 bean, and this is a point of importance, in view of the belief 

 that each species of I.eguminosse may have its own species of 

 bacteroid. 



It is especially the very young root-hairs, with extremely 

 delicate cell-walls, that are infected, and the first sign is the 

 appearance of a very brilliant colourless spot in the substance of 

 the cell-wall : sometimes it is common to two cell-walls of root- 

 hairs in contact, and not unfrequently one finds several root- 

 hairs all fastened together at the common point of infection. 

 This highly refringent spot is obviously the "bright spot" 

 referred to in the author's previous paper as the point of 

 infection from which the infecting filament takes origin. It 

 soon grows larger, and develops a long tubular process, which 

 grows down inside the root-hair, and invades the cortex, passing 

 across from cell to cell, as described in 1887. 



As a matter of fact, then, the "bright spot " is the point of 

 origin of the infecting filament ; and, as a matter of inference 

 from the experiments, it cannot but be developed from one 

 of the "bacteroids" or "gemmules" of the tuberc'es. This 

 attaches itself to the root-hair, fuses with and pierces the 

 delicate cellulose wall, and grows out into a hypha-like filament 

 at the expense of the cell contents. The further progress of 

 this filament has already been described in the author's memoir 

 in the Philos )phical Transactions for 1887. 



Researches were made during 1888 and 1889 with the object 

 of learning more about the conditions which rule the devclop- 

 rnent of the tubercles, and the relations of the organism to them. 

 The experiments seem to prove conclusively that the well being 

 of the organism of the tubercle and that of the pea or bean go 

 hand in hand. This ,of course is only so much evidence in 



favour of the view that we have here a case of symbiosis of the 

 closest kind, as expressed in the previous memoir. 



During the spring and summer of 1888 numerous experiments 

 were made with water-cultures with beans, allowed to germinate 

 in soil so as to be infected by the " germs " therein, as demon- 

 strated previously. Several dozens of such cultures were made, 

 and .'ome of them placed in the dark, others in the ordinary 

 light of the laboratory, and some in a well-lighted greenhouse. 

 Tables were prepared showing the number of leaves, living and 

 dead, the condition of the roots, the height of the stem, and so 

 forth, as recorded every week or so (or at shorter intervals) 

 when the plants were examined. It resulted that, when the 

 beans are in any way so interfered with that they do not assimi- 

 late more material than is necessary for the growth and im- 

 mediate requirements of the plant, the infecting organism either 

 gains no hold at all on the roots, or it forms only small tubercles 

 which are found to be very poor in " bacteroids ": in some cases 

 the starving plants began to develop tubercles, which never 

 became larger, and in which the infecting organism seemed to 

 be in abeyance. Whether this is due to the bacteroids being 

 developed in small quantities, or to the'r absorption into the 

 plant, is still a question. 



In these tubercles the chief difference was the paucity in 

 bacteroids, and the prominence of the branched filaments in the 

 cells. 



In the spring of this year (1889) the author started a series of 

 water-cultures of beans, infected artificially by placing the 

 contents of tubercles on their root-hairs, and kept the roots 

 oxygenated by passing a stream of air through the culture liquid 

 for twenty-four hours at intervals of a few days : here again the 

 increased growth of the plants — not compensated by increased 

 assimilation — seemed to cause the suppression of the tubercles, 

 or the formation of very poor ones only. These and similar 

 experiments lead to the conclusion that the organism which 

 induces the development of the tubercles is so closely adapted to 

 its conditions that comparatively slight disturbances of the 

 conditions of symbiosis affect its well-being : it is so dependent 

 on the roots of the Leguminossc, that anything which affects 

 their well-being affects it also. 



Some experiments with peas, which are now being tabulated, 

 may throw some light on the wider question which has been 

 raised of late, as to the alleged connection between the develop- 

 ment of these tubercles and the increase of nitrogen in Legu- 

 minous plants. Thirty-two peas were sown in separate pots of 

 silver-sand, or soil, in five batches of six each, and one of two, 

 and treated in various ways. 



The tubercles were developed on all but one of the plants, 

 except those in the completely sterilized media. The evidence 

 at present goes to show that the Leguminous plant gains nitrogen 

 by absorbing the nitrogenous substance of the bacteroids from the 

 tubercles ; that nitrogenous substances are thus brought by the 

 "bacteroids" ("gemmules") of the infecting organism of the 

 plant ; and that, finally, no satisfactory explanation seems forth- 

 coming as to how the organism obtains this nitrogen in certain 

 cases where no compounds of nitrogen have been added. At 

 any rate, if we regard the pot of sand and its pea as one system, 

 there is in some cases a distinct gain of nitrogen in the crop, and 

 in the sand at its roots. 



The author then refers to the literature since 1887, and reviews 

 two papers by Prazmowski which bear directly on these re- 

 searches. 



"To sum up, Prazmowski's account of the whole" matter 

 confirms that given to the Royal Society by the author in 1887, 

 excepting that he interprets the origin and nature of the bac- 

 teroids differently ; he regards them as produced froin the 

 contents of the filaments — as germ-like bodies developed in the 

 interior of the filaments, and not budded off from them. This 

 is hypothesis only, however, for the author expressly states (p. 

 253), ' Direct habe ich ihre Theilungen nicht gesehen, obgleich 

 ich mir die Miihe gab, sie in den verschiedensten Nahrmedien 

 und unter den verschiedensten iiusseren Bedingungen zu ziichten.* 

 He concludes they can only multiply in the still living pro- 

 toplasm. 



" As to the shapes of the bacteroids and tubercles, Prazmowski's 

 statements agree with those of previous observers, and he also 

 remarks the Plasmodium like appearance of the cell protoplasm 

 at certain stages, as noticed by myself. Some observations on 

 a possible spore-formation need not be dwelt upon, as he 

 recognized his mistake in a subsequent paper in 1889, 



