& . >O UNIVERSITY OF ILLINOIS Agricultural Experiment Station BULLETIN No. 202 IS SYMBIOSIS POSSIBLE BETWEEN LEGUME BACTERIA AND NON-LEGUME PLANTS? BY THOMAS J. BURRILL AND ROY HANSEN URBANA, ILLINOIS, JULY, 1917 CONTENTS OF BULLETIN No. 202 PAGE INTRODUCTION , 115 PAET I. THE OEGANISM '. 116 Isolation and Cultivation 116 Morphology 118 Cultural Characteristics 123 PAET II. CEOSS-INOCULATIONS: VABIETIES OP NODULE BAC- TEEIA -.125 Cross-Inoculation Investigations 125 Grouping by Serologieal Tests and by Cultural Differences 137 PAET III. HISTOLOGY OF THE NODULES OF THE LEGUMINOSAE . . 141 Origin of the Nodule 142 Structure of the Nodule 142 PAET IV. NON-LEGUMES SAID TO BE CONCEENED IN THE FIXA- TION OF ATMOSPHEEIC NITEOGEN 145 Historical 145 Ceanothus americanus 145 Cycas revoluta 148 Alnus, Elaeagnus, and Myrica 149 Conclusions 150 PAET V. ATTEMPTS TO DEVELOP A SYMBIOSIS BETWEEN LE- GUME BACTEEIA AND NON-LEGUME PLANTS 151 Evidence of Constancy or Change in the Organism 152 Experiments Attempting the Infection of Non-Legume Plants with Ps. radicicola 155 SUMMAE.Y 160 PAET VI. BIBLIOGEAPHIES 161 Symbiotic Nitrogen Fixation by Legumes 161 Non-Legume Boot Nodules 179 ILLUSTRATIONS PLATE I. Fig. 1. Ash-agar plate from bean. Fig. 2. Ash-agar plate from per- ennial pea II. Fig. 1. Ash-agar plate from pea. Fig. 2. Ash-agar plate from dyer's greenweed III. Fig. 1. Bacteroids from a very young nodule of pea. Fig. 2. Bac- teroids from young growing nodule of hairy vetch. Fig. 3. Bac- teroids from an older nodule of hairy vetch IV. Pseudomonas radicicola, showing polar flagellum : organisms from cow- pea, partridge pea, acacia, tick trefoil, and Japan clover V. Pseudomonas radicicola, showing polar flagellum : organisms from velvet bean, peanut, wild indigo, hog peanut, soybean; also B. subtilis in- troduced for comparison VI. Seedlings of partridge pea inoculated with bacteria from cowpea VII. Seedlings of cowpea inoculated with bacteria from partridge pea VIII. Seedlings of cowpea inoculated with bacteria from six species of Acacia IX. Seedlings of cowpea inoculated with organisms from partridge pea, tick trefoil, dyer's greenweed, Japan clov.er, velvet bean, cowpea, acacia, peanut, wild indigo X. Seedlings of alfalfa grown after Garman 's method, showing inoculation by several cultures XL Fig. 1. Longitudinal section of a nodule of red clover. Fig. 2. Cross- section of a similar nodule of red clover XII. Fig. 1. Cross-section thru the meristem region of a nodule of hairy vetch. Fig. 2. Cross-section thru the same nodule some distance back from the meristem. Fig. 3. Infection threads in the- cortex cells of a nodule of red clover XIII. Fig. 1. Young infected cells of a nodule of hairy vetch. Fig. 2. Bac- teroidal cells of red clover in a well advanced but growing nodule XIV. Eoot nodules of Ceanothus americanus XV. Fig. 1. Longitudinal section of a Ceanothus americanus nodule. Fig. 2. Cross-section thru a similar nodule of Ceanothus americanus XVI. Fig. 1. Parasitized cells of a Ceanothus americanus nodule. Figs. 2 and 3. Same more highly magnified XVII. Experiment VIII : Morning-glory plants inoculated with sweet-clover bacteria FOREWORD This bulletin reports the last work of Thomas Jonathan Bur rill, who in 1880, thru studies of pear blight, first experimentally proved the fact that plant diseases are sometimes caused by bacterial invasion. Symbiotic relationships early attracted the attention of Dr. Bur- rill, especially the relationship existing between certain nitrogen- gathering bacteria and legumes. In those days every discovery gave rise to new and fundamental questions, and the query whether such relation is necessarily confined to legumes was always in his mind, and was put aside only by the urgency of pressing duties. When after retirement from active service, opportunity came to Dr. Burrill for following his inclination, his attention at once reverted to the old-time problem, and he fitted up a laboratory and employed an assistant for its study. Here he devoted the last three years of his life, and here the call came suddenly on April 14, 1916, forty-eight years to a month after his coming to the University of Illinois. Especial credit is due the junior author for his faithful and hope- fully successful attempt at accurately reporting the work as planned by his chief, and so far as is humanly possible correctly interpreting his ideas and convictions. In this difficult task Mr. Hansen has been aided by Dr. A. L. Whiting with some special knowledge of the technical material in- volved, and by Professor C. F. Hottes, for a quarter of a century Dr. Burrill 's close associate, who has read the manuscript with the view of insuring that so far as possible the spirit and thought of the pioneer investigator is expressed. E. DAVENPORT Director IS SYMBIOSIS POSSIBLE BETWEEN LEGUME BACTERIA AND NON-LEGUME PLANTS? By THOMAS J. BURRILL, PROFESSOR OF BOTANY, EMERITUS, AND EOY HANSEN, ASSISTANT IN NITROGEN-FIXATION RESEARCH INTRODUCTION The work reported in this bulletin deals with an attempt to develop a symbiosis between legume bacteria and non-legume plants similar to that which exists between legume bacteria (Pseudomonas radicicola) and legume plants. Since the demonstration, in 1886, by Hellriegel and Wilfarth of the symbiotic fixation of atmospheric nitrogen by legume plants and cer- tain microorganisms, no crop rotation has been considered rational that does not include a liberal use of legumes. The importance of this discovery to agriculture is generally appreciated. That it is applicable thruout the world makes it of especial value to mankind. The benefit that would result could other ordinary farm crops be enabled to utilize atmospheric nitrogen would be inestimable; hence the importance of any success in this direction. In attempting to study this question it was fully realized that success might not be attained, but that it was in the realm of possibilities. It was nearly a quarter of a century ago that the first work was done under the direction of the senior author. Since that time a few attempts have been made by other workers to grow legume bacteria on mustard and grasses, but with negative results. In returning to this problem, the authors found it necessary at first to spend considerable time in acquiring an intimate acquaintance with the organism concerned, especially in regard to its cultivation and identification. Attention was given to the special adaptations, or varieties, of the symbiotic bacteria in order to learn, first, whether these adaptations were constant or subject to change ; and second, what factors were responsible for their existence. Histological studies of the nodule were undertaken with the view of learning something of the relations existing between the two symbionts. The nodules of certain non-legume plants (Ceanothus, Cycas, Elaeagnus, etc.), said to be concerned in the fixation of atmospheric nitrogen, were given some attention in the hope that perhaps here lay a start. Cross-inoculations of importance and interest were found and are reported as a part of this contribution. Some preliminary trials were made attempting the inoculation of non-legume plants with the legume organism. 115 116 BULLETIN No. 202 [July, Part I. THE ORGANISM ISOLATION AND CULTIVATION Media. Pseudomonas radicicola was cultivated on many kinds of media differing widely in composition, and it was found that it would thrive on most of them. For plating out, Harrison and Barlow's wood-ash agar was usually used, as it gave more uniform results. Many media were unsuitable for plating, yet permitted growth upon agar slants. A list of media employed in these experiments for cultivating Ps. radicicola, together with the composition and reaction of each, is given in Table 1. TABLE 1. COMPOSITION AND KEACTION OF MEDIA USED IN CULTIVATING Pseudomonas radicicola Labora- tory No. Medium Composition Reaction* 100 Wood ash (Harrison and Barlow) Wood-ash extract (15 gins, ashes to 1 liter tap water) 1000 cc. Saccharose 10 gms. Monopotassium phosphate 3 gms. Not changed; usually -(-7 to -4-10 to phenol - phthalein 101 Synthetic (Fred) Distilled water 1000 cc. Dextrose 20 gms. Monopotassium phosphate 1 gm. Magnesium sulfate .1 gm. Sodium chlorid Trace Ferrous sulfate Manganous sulfate Calcium chlorid " Titrate to +10 102 Mannite (Ashby) Distilled water 1000 ce. Mannite 20 gms. Dipotassium phosphate .2 gm. Magnesium sulfate .2 gm. Sodium chlorid .2 gm. Calcium sulfate .1 gm. Calcium carbonate 5 gms. Not changed 103 Synthetic (Spratt) Distilled water 100 cc. Cane sugar ' 1 gm. Dipotassium phosphate .5 gm. Magnesium sulfate .02 gm. Calcium carbonate .1 gm. Titrate to +10 104 Asparaginate (Conn) Distilled water 1000 cc. Sodium asparaginate 1 gm. Dextrose 1 gm. Magnesium sulfate .2 gm. Ammonium phosphate" 1.5 gms. Calcium chlorid .1 gm. Potassium chlorid .1 gm. Ferric chlorid Trace Not changed; usually +6 to +8 Fuller's scale in all cases. b Used in place of mono-ammonium phosphate. 1917] POSSIBLE SYMBIOSIS BETWEEN LEGUME BACTERIA AND NON-LEGUMES 117 TABLE 1. Continued Labora- tory No. Medium Composition Eeaction 105 Beef broth Tap water 1000 cc. Witte's peptone 10 gms. Beef extract (Liebig's) 5 gms. Titrate to +10 106 Legume extract, using bean plant Extract of bean plant (Heat 100 gms. roots and stems in 1 liter tap water y 2 hour at 60 C.) 1000 cc. Cane sugar 20 gms. Titrate to +10 107 Bean-extract peptone Same as 106, plus 1 percent peptone (Witte's) Titrate to +10 108 Legume extract, using sweet clover Sweet-clover extract 1000 ce. Cane sugar 20 gms. Titrate to +10 109 Sweet-clover - extract pep- tone Same as 108, plus 1 percent peptone Titrate to +10 110 Tomato infusion Tomato extract (100 gms. plant substance to 1 liter water) 1000 cc. Cane sugar 20 gms. Titrate to -flO 111 Tomato-infu- sion peptone Same as 110, plus 1 percent peptone Titrate to -f 10 200 Wood-ash agar Same as 100, plus 1 percent agar Not changed 201 Synthetic agar (Fred) Same as 101, plus 1 percent agar Titrate to +10 202 Mannite agar (Ashby) Same as 102, plus 1 percent agar Not changed 203 Synthetic agar (Spratt) Same as 103, plus 1 percent agar Titrate to +10 204 Asparaginate agar Same as 104, plus 1 percent agar Not changed 205 Beef -broth agar Same at 105, plus 1 percent agar Titrate to +10 206 Bean-extract agar Same as 106, plus 1 percent agar Titrate to +10 207 Bean-extract- peptone agar Same as 107, plus 1 percent agar Titrate to +10 208 Sweet-clover-ex- tract agar Same as 108, plus 1 percent agar Titrate to +10 209 Sweet-clover-ex- tract-peptone agar Same as 109, plus 1 percent agar Titrate to +10 210 Tomato-extract agar Same as 110, plus 1 percent agar Titrate to +10 211 Tomato-extract- peptone agar Same as 111, plus 1 percent agar Titrate to 4-10 300 Wood-ash gelatin Same as 100, plus 12 percent gelatin Not changed 305 Beef-broth gelatin Same as 105, plus 12 percent gelatin Titrate to -f 10 420 Potato slant 421 Tomato-stem slant Fresh young tomato stems in distilled water 118 BULLETIN No. 202 Isolation. In isolating the organism the following method adapted from that of Harrison and Barlow 32 " was used: Where choice is possible select a medium sized nodule appearing young and sound. In cutting it off leave two or three millimeters of the root on both sides of the nodule to permit handling it with forceps. Wash carefully, rinse in distilled water, and drop into a sterilizing fluid made as follows : Distilled water 500 cc. Bichlorid of mercury 1 gin. Hydroclorie acid (C. P.) 2.5 cc. Shake the nodule violently in this solution for one or two minutes, after which wash it three times with sterile distilled water. Then cover with about 1 cc. of sterile distilled water and crush with a heavy glass rod, previously flamed and cooled. Pour two or three drops of the cloudy suspension into a test tube of ash agar b at 45 C. Inoculate a second tube of the agar with five loops from the first, and pour plates. When a large nodule is used, inoculate the first tube with five loops of the suspension, and inoculate the second tube with five loops from the first, and pour plates: Only two plates are poured ; a third was found unnecessary. Incubate plates at from 20 to 25 C. Replating is usually unnecessary, altho it is a safe practice with ques- tionable plates. If the sterilization and washing are carefully done, foreign organisms seldom appear. Cultivation. For keeping stock cultures ash agar was used. Transfers were made once a month, tho cultures may easily be kept six weeks or two months between transfers. Plate colonies should be large enough for transfer in six to fourteen days, depending upon the host plant used and other conditions. MORPHOLOGY Agar Colonies. In general the colonies appearing on agar plates may be divided into two types, buried and surface colonies. Buried colonies are small and submerged, most frequently lens, or spindle shaped, with smooth and even edges. They are quite opaque, granular in structure, and in color are cream to a chalk white. They increase slowly in size, eventually appearing on the surface of the agar as surface colonies, when the growth becomes rapid. The Superior figures are used to indicate the literature citations having special reference to this work which are given in the bibliographies. "Obtain ashes from thoroly burned hard wood and run thru a fine sieve. (Little difference was found in different lots of ashes.) "Use 15 grams to one liter of tap water and bring to a boil over a free flame, stirring at intervals. Allow solution to stand from five to ten minutes, then filter thru a double filter. To one liter of ash extract add 10 grams of saccharose, 3 grams of KH 2 PO<, and 10 grams of agar. Autoclave for fifteen minutes, filter thru absorbent cotton, and proceed as with other media. The reaction usually was -f7 to +10 (Fuller's scale) to phenol- phthalein, and was never changed. 1017] POSSIBLE SYMBIOSIS BETWEEN LEGUME &ACTERIA AND NON-LEGUMES 119 lens colonies, however, remain visible for many days in the center of the new growth. Surface colonies originate at or near the surface of the agar or develop from buried colonies. They are drop-form, watery, mucilagi- nous (in appearance, tho not always to the touch), gray-white to pearly white in color, glistening, and semitranslucent to opaque. The edges are smooth and even. Under the low power the interior is granu- lar. They frequently attain considerable size, a centimeter or more in diameter. Plates made direct from the nodule lack uniformity to a marked degree. The undiluted plate (first plate) begins to show a few colonies in two to four days. These colonies become extremely large in a very short time, their rapid growth being due to small pieces of nodule tissue or to clumps of bacteria carried over into the agar (see Plate I). In five or six days numerous colonies begin to make their appearance, most of them as submerged colonies, which later grow to the surface. The dilution-plate (second-plate) colonies are always extremely slow in growth. Generally colonies are large enough for transfer in six to fourteen days, tho plates should not be discarded for two or even three weeks. The rate of growth of colonies also varies with the organisms of different nodules (see Plate II). Among the fast growers are the organisms from the pea (Pisum), vetch (Vicia), lentil (Lens), sweet pea (Lafhyrus), bean (Phaseolus) , lupine (Lupinus), wild bean (Strophostyles) , clover (Trifolium), sweet clover (Melilotus), alfalfa (Medicago), and fenugreek (Trigonella). The organisms appreciably slower in growth are those from the cowpea (Vigna), Japan clover (Lespedeza), tick trefoil (Desmodium), acacia (Acacia), partridge pea (Cassia), false indigo (Baptisia), dyer's greenweed (Genista), peanut (Arachis), soybean (Glycine), and hog peanut (Amphicarpa) . The Bacteria. The life cycle of Pseudomonas radicicola from the soil thru the nodule and back to the soil is clouded in doubt because of the extreme variability of the organism under apparently the same conditions. While it has been isolated from soil (see Lip- man 41 42 ), there is no clue to the form in which it existed in the soil. Observation of cowpea nodules showed that in the very young nodules there is considerable variation in size and shape of the organ- isms. Many of the small, oval forms, the swarmers described by Beyerinck, 9 are found. These forms and the normal rods predomi- nate. Large club-shaped bacteroids are frequent; the characteristic branched forms are not so numerous. The bacteroids are best demon- strated when the young nodule is just beginning to show a reddish interior. At this stage they are extremely large and contain the maxi- mum staining substance (see Plate III). The characteristic X and Y forms occur in great numbers ; they show considerable vacuolation and unevenness in staining, especially when stained with carbol- fuchsin. 120 BULLETIN No. 202 PLATE I Fig. 1. Ash-agar plate from bean (Pkaseolus vulgaris}, showing giant col- onies in a thickly seeded plate Fig. 2. Ash-agar plate from perennial pea (Lathyrus latifolius) ; the clear spaces are due to sterilizing fluid carried over with pieces of nodule tissue 1917] POSSIBLE SYMBIOSIS BETWEEN LEGUME BACTERIA AND NON-LEGUMES 121 In the old, decomposing nodule the bacteroids are extremely vacuolated and ghost-like, showing small, oval, deep-staining bodies within. The inference is that these bodies are motile swarmers, which later free themselves from the ghost-like capsules, rather than bud off, as has been described by some writers. Frequently the swollen rods have a beaded appearance with unstained bands or areas. A few motile rods may sometimes be seen in hanging drops in this stage, and sometimes a bacteroid is seen to oscillate as tho swung about by some propelling force in one end. Division of the bacteroids into bacilli, as represented by Dawson, 23 may also occur. When first plated out, the young colonies consist of small rods which show considerable variation in length. No bacteroids are pres- ent, tho the rods are sometimes slightly club-shaped and sometimes show vacuolation. However, they never attain the size of bacteroids. With frequent transfers the rods become quite uniform in size and slain deeply and evenly, especially with aniline-gentian-violet. In very old cultures (three months on ash agar, without transfer) the small, oval swarmers and the normal rods predominate, tho a few club-shaped and a few branched bacteroids are found. The bacteroids produced upon artificial media* are never so large nor so numerous as those seen in mounts direct from a young nodule. Staining. The organisms do not stain well with ordinary aniline stains. Carbol-fuchsin and aniline-gentian-violet (used steaming) are the most satisfactory stains. Tho carbol-fuchsin was preferred, aniline- gentian-violet stains were always used as checks, because the former slain accents the vacuolated appearance, particularly in bacteroids. Carbol-fuchsin is especially useful in staining bacteroids direct from the nodule and also old agar cultures. Kiskalt's amyl-gram stain, described by Harrison and Barlow, 32 is useful since the amyl alcohol clears up the field, leaving the bacteria stained, tho not so intensely. This stain, however, should not be considered a means of identifying Ps. radicicola. Bacteroids. While Ps. radicicola produces no spores, it produces bacteroids which are very evidently more resistant than the normal rods. Unfavorable conditions, such as unsuitable media, infrequent transfer, or addition of caffein to the medium, cause their appearance. This is in accord with what takes place in the nodule. In the growing nodule, when development is most rapid, the bacteroids are at their maximum ; they enable the organisms to multiply rapidly in spite of the resistance offered by the plant cells. Transferred to favorable media from this stage the normal uniform bacilli are produced. The bacteroid, then, must be regarded as a normal and a very necessary From the writers' observations this is equally true of the bacteroids produced by adding caffein to a legume-extract-agar medium, according to the method of Z'ipfel 37 and Fred." 122 BULLETIN No. 202 [July, stage in the life of the organism. Its significance in the actual fixation of nitrogen, however, is pure speculation. Motility. The motility of the organism is best seen in young agar- slant cultures, twenty-four to forty-eight hours old. The bacteria dart about with amazing rapidity, now tumbling end over end, now spinning violently on the shorter axis, and then sweeping across the field in a darting, jerky course. Flagella. Owing to the gum or slime produced by the organism, the demonstration of flagella is especially difficult. The lack of agree- ment among investigators as to the number is shown in Table 2. The organisms reported by these investigators were all the most abundant producers of gum. TABLE 2. RESULTS OF PREVIOUS WORK UPON FLAGELLA STAINS Investigator Source of organism Flagella Remarks Beyerinck 1888 One polar flagellum Inferred during slow motility and not seen Smith, R. G. 20 1899 Exceedingly thin, single, terminal flagel- lum about 2 microns long and bearing up- on the distal end a tuft, like the lash of a whip One photomicrograph Harrison and Barlow 1907 Hairy vetch (Vicia villosa} . Perennial pea (Lathyrus sativus*) Bean (Phaseolus vulgaris) . Single polar flagellum Several figures; mu- cilage, or negative method, by which slime is stained leav- i n g flagella u n- stained; discredited by Kellerman De Eossi 30 1907 Broad bean (Vicia faba) Bacillus No figures; describes white, non-liquefy- ing, non-infectuous intruder which has a polar flagellum De Rossi 33 1909 White clover (Trifolium repens} and other clovers 8 to 10 flagella; peri- triehic One photomicrograph of Trifolium repens; very good Kellerman 1912 Garden pea (Pisum sativum) Lima bean (Phaseolus lunatus) Alfalfa (Medicago sativa) Flagella fairly nu- merous; peritrichic Three photomicro- graphs, none of which is convincing Zipfel 1912 Numerous flagella; peritrichie No figures Prucha 43 1915 Canada field pea (Pisum sativum arvense) Peritrichic ; largest number observed was six, but there may be more No figures 1917] POSSIBLE SYMBIOSIS BETWEEN LEGUME BACTERIA AND NON-LEGUMES 123 Organisms from red clover (Trifolium pratense), broad bean (Vicia faba), hairy vetch (Vicia vttlosa), common bean (PJiaseolus vulgaris), sweet clover (Melilotus alba), alfalfa (Medicago sativa), field pea (Pisum arvense), and sweet pea (Lafhyrus odoratus) were stained for flagella, using several methods, but the gum stained so heavily that none could be seen. The production of gum by the organism, as will be shown later, depends more upon the plant species from which it is isolated than upon the culture medium. Attention was then turned to the organisms making less vigorous growth, which produce less gum. Successful stains were made of the organisms from cowpea (Vigna sinensis), tick trefoil (Desmodium canescens), dyer's greenweed (Genista tinctoria), velvet bean (Mucuna utilis), peanut (Arachis liypogoea), wild indigo (Baptisia tinctoria), Japan clover (Lespedeza striata), acacia (Acacia floribunda), partridge pea (Cassia chamaecrista) * soybean (iGlycine tiispida), and hog pea- nut (Amphicarpa monoica). Loeffler's method of staining was used. The mordant" was made up as follows : Solution of tannin (20 percent in water) 10 parts Saturated (cold) aqueous solution of ferrous sulf ate 5 parts Saturated alcoholic solution of basic f uchsin ... 1 part Transfer the organisms successively several times upon ash agar to hasten the growth. With a platinum needle transfer some of the organisms from the edge of a transfer two or three days old to a small drop "of sterile water upon a clean cover slip. Spread slowly and care- fully (only a few strokes are necessary), and allow to dry. Cover well with a mordant, bring to a steam, and allow to stand about one minute. Wash carefully with distilled water and apply carbol-fuchsin, bring to a steam, and again let stand one minute. In examining the slide, look especially near the edges of the smear and close to the "drifts" of bacteria. Slime and stain deposits fre- quently interfere, tho not seriously. The organism has a single polar flagellum (see Plates IV and V). It was noted that the flagellum is rarely attached at the end, but rather at a corner. CULTURAL CHARACTERISTICS Ps. radicicola will grow between and 50 C. The optimum temperature is 25 to 28 C., tho it will grow well at room tem- perature, or 20 to 25C. The organism is aerobic. The diffused light The organisms of these nine plants comprize a single group, i. e., they are indentical, as will be shown later. Isolations, however, were made from the host plants as named. Actually, then, but three distinct varieties were stained Vigna, Glycine, and AmpJiicarpa. "Filter the ingredients separately and mix in the order given. Filter direct' upon the cover slip. The mordant is best used fresh. 124 . BULLETIN No. 202 [Jul>i, of the laboratory is not harmful. Even exposure to direct sunlight for several months without transfer did not kill organisms when grown upon favorable media with precautions to prevent evaporation. Under such conditions a temperature of 47 C. in the flask was reached with the thermometer shaded. Slight alkalinity to +20 to +25 acid (Fuller's scale) with phenolphthalein is tolerated; neutral to -(-10 is best. Growth is generally better in gelatin or agar media than in liquid media of the same composition. In an agar stab a typical drop-form colony is produced at the sur- face. A thin, gray growth follows the line of stab. MaltoSe as a source of carbon has little if any advantage over saccharose or dextrose. Mannite is also suitable as a source of carbon. In standard beef broth the growth of the organism is slow. The liquid becomes cloudy, a gray-white ring is formed, and a thin mem- brane covers the surface. Later a flocculent precipitate settles to the bottom of the tube. In standard beef-broth gelatin the growth of the organism is at first funnel-shaped and then stratiform. The gelatin slowly liquefies, the process sometimes requiring two or three months for completion. In gelatin stabs the growth sometimes seals over the stab with a drop-form growth and liquefaction does not occur. If inoculated tubes are kept for several weeks at a temperature just allowing the gelatin to remain liquid, upon cooling it will be found that the gelatin refuses to solidify, whereas the gelatin in uninoculated check tubes does solidify. The enzyme causing liquefaction is present. On ash-agar plates the presence of Penicillium glaucum, which occasionally intruded, seemed to benefit the colonies of Ps. radicicola that were in close proximity. Ash agar upon which Penicillium glaucum had been allowed to groAv for two weeks and which had then been sterilized and filtered, had a noticeable advantage over untreated ash agar, especially with the slower growing organisms, such as those of Vigna, Glycine, and Genista. Ti0J, PLATE II Fig. 1. Ash-agar plate from pea (Pisum sativum}, seven days old Fig. 2. Ash-agar plate from dyer's greenweed (Genista tinctoria), twenty- five days old V x "2 '3 X 43 a=>. co / * r ^ . : O O i ? M > o > r_ ^. .- ass mmm rH N CO bb bb bio N ' f * % t , * > J -* ~x* I ,,.* ,- / * - -* - .t -w ^ r> i S? 10 * o ^S 93 I s ' rO we >bo s M CO ll ' < ^ / 1: ** . 111 v r^ ^ 1917] POSSIBLE SYMBIOSIS BETWEEN LEGUME BACTERIA AND NON-LEGUMES 125 Part II. CROSS-INOCULATION: VARIETIES OF NODULE BACTERIA CROSS-INOCULATION INVESTIGATIONS It was early recognized that certain legumes require one specific organism for inoculation. For example, to inoculate soybeans it had been found necessary to import soil upon which soybeans had grown, as the bacteria from other legumes were not capable of causing infec- tion. A few cross-inoculations which occur under field conditions were also early recognized. The bacteria of alfalfa and sweet clover* were known to be identical, as were those of the cowpea and partridge pea. A third group the bacteria of which were known to be inter- changeable included pea, vetch, sweet pea, and lentil. Several investigators, notably Laurent, 14 Maze, 21 Moore, 28 and Kellerman, 36 claimed to have produced cross-inoculations which do not occur naturally. Little credence can be given these claims, however, since these men apparently did not fully appreciate what has been frequently referred to as the ubiquity of Ps. radicicola. No doubt their technic was at fault. Methods Used in Cross-Inoculation Work. For testing cross-in- oculations bacteria were isolated from as many genera and species of legumes, both wild and cultivated, as could be obtained. Great care was taken in their isolation and in the maintenance of purity. Two methods of testing crosses were used the pot-culture method and the agar test-tube method of Garman. 38 In the pot-culture method, plants were grown in one-gallon pots of limed white quartz sand watered with a nutrient solution less nitrogen, as described by Hopkins and Pettit. b The sand was not sterilized, as it was dry and clean, and sterile so far as legume bacteria were concerned, as proven by the record of the check pots. The pots were washed clean and exposed to sunlight in the greenhouse a week before using, being turned several times. A number of dry, clean pots were always on hand so that no "Hopkins. 2 * Trom Hopkins and Pettit Laboratory Manual for Soil Fertility, page 34. Solution No. 1. Nitrogen: Dissolve 80 grams of ammonium nitrate in 2500 cc. of distilled water. Solution No. 2. Phosphorus: Dissolve 25 grams of mono-calcium phos- phate in 2500 cc. of ammonia-free water. Solution No. 3. Potassium: Dissolve 50 grams of potassium sulfate in 2500 cc. of ammonia-free water. Solution No. 4. Magnesium: Dissolve 20 grams of magnesium sulfate in 2500 cc. of ammonia-free water. Solution No. 5. Iron: Dissolve .1 gram ferric chlorid in 250 cc. of ammonia-free water. Use 10 cc. each of Solutions Nos. 1, 2, 3, and 4, and 1 cc. of Solution No. 5 per liter of water. When, nitrogen is omitted the fact is so stated. 126 BULLETIN No. 202 [July, 1U17] POSSIBLE SYMBIOSIS BETWEEN LEGUME BACTERIA AND NON-LEGUMES 127 loss of time resulted when organisms were to be tested. The seeds were sterilized by shaking them violently in Harrison and Barlow's steril- izing fluid, previously described, allowing them to remain in the fluid for ten minutes, and then washing in distilled water. Usually five seeds were planted in a pot. Inoculation was made at the time of planting by adding the contents of an agar slant mixed with sterile distilled water. One pot in each four was left uninoculated as a check. Occasionally scattered nodules did appear on checks and in pots which when repeated gave negative results. The use of open pots in a greenhouse frequented by many people, together with the presence of occasional insects, etc., cannot but result in some chance inoculations. A chance inoculation, however, is easily distinguished from a true one, for in the former case the nodules are few in number and widely scat- tered, whereas in a true inoculation the nodules are numerous and clustered in a mass about the tap root. This pot-culture method was used for growing plants with large seeds, such as Vigna, Glycine, P'isum, Vicia, Lathyrus, and Phaseolus. In the second method used, that of Garman, 38 seeds were planted in test-tubes (6" x %") containing a medium composed of .65 percent agar in distilled water. No nutrients were added. The agar was inoculated at 42 to 45C. Seeds (usually three to a tube), sterilized as before, were dropped upon the agar and set apart with a flamed platinum needle. Generally the nodules resulting were not numerous, but where the seeds germinated well, results were always positive and dependable. This method of testing crosses is especially adapted to smaller seeds, such as Melilotus, Medicago, and Trifolium. Large seeds give trouble, as they are difficult to sterilize. Vigna X Cassia. The inoculation of the cowpea by bacteria from the partridge pea was first reported by Hopkins. 34 In the other cross- inoculations mentioned above (alfalfa and sweet clover; pea, sweet pea, vetch, and lentil), the plants having a common organism stand in close botanical relationship," while Vigna sinensis and Cassia cliamaecrista are widely separated. Moreover, the former is a plant introduced from Asia, while the latter is a native. The first cross-inoculation experiments in these investigations were conducted with the partridge pea (Cassia chamaecrista) , inocu- lating it as shown in Table 3. Partridge-pea seeds and nodules were obtained from plants found upon virgin prairie in a wild locality where in all probability cowpeas had never been grown. Cultures were obtained from cowpea nodules grown in the greenhouse. Thus the sources of the organisms were wide apart. The seeds were planted on October 16, 1915, three in each pot ; the plants were examined and photographed on November 19 (see Plate VI) . The results appear in Table 3. The number of nodules reported Engler und Prantl: Die Naturlichen Pflanzenfamilien, III. 128 BULLETIN No. 202 \Jubj, 1917] POSSIBLE SYMBIOSIS BETWEEN LEGUME BACTERIA AND NON-LEGUMES 129 may be somewhat low, as it is difficult to count the smaller nodules. The checks were examined with great care and found to be free from nodules. TABLE 3. PARTRIDGE PEA x COWPEA (Cassia chamaccrista X sinensis} Pot No. Plant No. of plants Source of inoculation Nodules Besults + or- 4593 4594 4595 4596 Partridge pea II II tt it it ii 3 3 3 3 Partridge pea No. 4608 " "No. 4609 " "No. 4611 "No. 4613 17,15, 9 3,11, 7 5, 5, 5 3, 3,11 4597 4598 4599 4600 Partridge pea a tt it it 3 3 3 3 Check it it it 0, 0, 0, 0, 0, 0, 0, 0, 4601 4602 4603 4604' Partridge pea it it it it it 3 3 3 3 Cowpea No. 4615 " No. 4617 " No. 4619 " No. 4621 12, 3, 8 8,10, 7 7, 8, 6 0, 0, "For some unknown reason the plants in this pot produced no nodules. The reciprocal was then tried. Five seeds of cowpea were planted in each pot and inoculations made as shown in Table 4. The seeds were planted on November 29, 1915 ; the plants were examined and photographed on January 3, 1916 (see Plate VII). The results are also shown in Table 4. TABLE 4. COWPEA x PARTRIDGE PEA (Vigna sinensis x Cassia chamaecrista) Pot No. Plant No. of plants Source of inoculation" Nodules Results -f- or 5023 5024 5025 5026 Cowpea ii a 5 5 5 5 Cowpea Nos. 4398 and 5042 " Nos. 4614 " 5039 ' Nos. 4616 : ' 5040 ' Nos. 4618 " 5041 Abundant ii + + 5027 5028 5029 5030 Cowpea it 5 5 5 5 Cheek None 5031 5032 5033 5034 Cowpea a 5 5 5 5 Partridge pea Nos. 4605 and 5035 " " Nos. 4607 " 5036 " Nos. 4610 " 5037 " " Nos. 4612 " 5038 Abundant 4- + Vigna X Acacia. Great interest had been taken in some prelimi- nary trials which had given evidence that a cross exists between cowpea and acacia. Accordingly cowpea plants were inoculated with cultures from six species of Acacia, and later with a culture from u seventh. These were Acacia armata, floribunda, linifolia, longifolia, semperflora, and a species the nodules of which had been received from 130 BULLETIN No. 202 [July, fcC 1917] POSSIBLE SYMBIOSIS BETWEKN LEGUME BACTERIA AND NON-LEGUMES 131 California but of which nothing else was known except that it is an ornamental tree. Cultures of the first five species were obtained from nodules of plants grown in the horticultural greenhouse on this cam- pus. Cultures from a seventh species, Acacia melanoxylon, which was later grown in this greenhouse, behaved exactly like the other six. Seeds were planted on January 13, 1916; the plants were examined and photographed on February 21 (see Plate VIII). The results are shown in Table 5. TABLE 5. COWPEA x ACACIA (Vigna sinensis X Acacia} Pot No. Plant No. of plants Source of inoculation Nodules Results -for 5464 5465 5466 Cowpea 5 5 5 Acacia armata Nos. 5578 and 5649 Acacia floribunda Nos. 5579 and 5650 Acacia linifolia Nos. 5580 and 5651 Abundant -- 5467 5468 5469 5470 Cowpea tt it 5 5 5 5 Check None 5471 5472 5473 Cowpea 5 5 5 Acacia longifolia Nos. 5581 and 5652 Acacia semperflora Nos. 5582 and 5653 Acacia ? (from California) Nos. 5583 and 5654 Abundant + + Cowpea X Several Generic Groups. Tests made from time to time with the cowpea had shown that infection could be produced with bacteria from eight different generic groups besides the cowpea organ- ism. An experiment was then conducted to bring together the results of these previous trials. The results are given in Table 6. TABLE 6. COWPEA ( Vigna sinensis) x SEVERAL GENERIC GROUPS Pot No. Plant No. of plants Source of inoculation Nodules Results -f-or- 6309 Cowpea 5 Check None 6310 5 Acacia (Acacia melanoxylon) Abundant + 6311 5 Lead plant (Amorpha canescens) None 6312 5 Hog peanut (Amphicarpa monoica) Several 6313 5 Check Several 6314 5 Peanut (Arachis hypogoea) Abundant -- 6315 5 Wild indigo (Baptisia tinctoria) Abundant -- 6316 5 Partridge pea (Cassia chamaecrista) Abundant 6317 5 Check None 6318 5 Tick trefoil (Desmodium canescens) Abundant + 6319 5 Dyer's greenweed (Genista tinctoria) Abundant 6320 5 Japan clover (Lespedeza striata) Abundant + 6321 5 Check None 6322 5 Common locust (Eohinia pseudo- acacia) Several 6323 5 Velvet bean (Mucuna utilis) Abundant + 6324 5 Cowpea (Vigna sinensis) Abundant 132 BULLETIN No. 202 [July, PLATE IX Seedlings of cowpea (Vigna sinensis) inoculated as follows: A. Partridge pea (Cassia cliamaecrista) ; B. Tick trefoil (Desmodium canescens)', C. Dyer's greenweed (Genista tinctoria) ; D. Check; E. Japan clover (Lespedeza striata) ; F. Velvet bean (Mucuna utilis) ; G. Cowpea (Vigna sinensis); H. Check; I. Acacia (Acacia melanoxylon) ; J. Peanut (Arachis hypogoea) ; K. Wild indigo (Baptisia tinctoria) ; L. Check POSSIBLE SYMBIOSIS BETWEEN LEGUME BACTERIA AND NON-LEGUMES In Plate IX two plants from each pot are shown; those which were negative have been omitted. The results confirmed those of the earlier tests. The plants in three of the negative pots (those crossed with Amphicarpa, Robinia, and a check) had several nodules, but these were no doubt accidental as they were very scattered. In a pre- vious trial, inoculations with Amphicarpa and Robinia had both given negative results. Lens X Several Generic Groups, Another set of similar experi- ments is of interest. Lentils were planted on March 17, 1916, and inoculated with bacteria from several generic groups. The seedlings were examined on April 14. The results are shown in Table 7. TABLE 7. LENTIL (Lens esculenta) x SEVERAL GENERIC GROUPS Pot No. Plant No. of plants Source of inoculation Nodules Results -for- 6386 Lentil 3 Scarlet runner bean (Phaseolus multi- florus) None 6387 > 3 Common bean (Phaseolus vulgaris) None 6388 > 3 Trailing wild bean (Strophostyles hel- vola) None 6389 i 3 Perennial pea (Lathyrus latifolius) Abundant + 6390 i 3 Common garden pea (Pisum sativum) Abundant 6391 . 3 Field pea (Piswm arvense) Abundant -)- 6392 i 3 Broad bean (Vicia faba) Abundant + 6393 3 Check None In a similar experiment with seedlings of Trigonella foenum- graecum, it was found that they could be infected with the bacteria from Melilotus alba and Medicago sativa. In fact, many inoculation trials were made with all available bacteria. Many results were nega- tive, as is shown in Table 9. Tests with Garman's Method. The cross-inoculation trials made by Garman's method together with the results obtained are shown in Table 8. Four inoculated tubes and one check were used for each cul- ture tested. In Plate X are shown seven cultures tested upon Medicago sativa (only one tube of each of the seven series is shown) together with two checks. In all, this photograph represents thirty-five tubes, twenty-eight inoculated and seven uninoculated. Results of Cross-Inoculation Trials. Table 9 gives in full the results of the cross-inoculation experiments conducted. All available cultures were tried upon seedlings of Anthyllis vulneraria and Mimosa pudica, but no nodules were produced. It is assumed that the organ- isms of these plants are distinct from any of those used. 134 BULLETIN No. 202 11? O *S 11 III II *<3 ^ ^ 1 1 III II . lit "* o''^ ^ < 53 1 i KB f JaSis 1 1 I I 1 ! ! +++++ i + OQ w i3? ^ 1 5 o 5 o c4 2 1 i "3 1 ] 1 1 ,1 I +++++ i + w p H *S s CO * "fc O x-N a ^ F *(J ^i Q s 3 o "f* 1 1 III +++++ i + tM v.x'^' c3 ^ ^^^ co tS y ATION 1 1/5 si 4 ? + 1 ++++++ 1 ++] M 1 1 + g H-S-_ S IH i W < CO oa Q o 53 2 ^ Hi H V Q ^ eS G O ^** ^i f od R a .2 liiil'lill^llllii^ll. .2 E S *~ 'S s _> A rfi ^ 1 I 1 i 1 1 -1 H P3 *^ 53 ^3 S^j si S ^5 ^j 7 ^ 5^ GO ^- 5S ^ r^ ? ^^1*" ^ ?^ S ^ Sj**^* to 5^ 9 03 "*? O i^ .C* "*"* S ** r* S S S5j ^. ^ . S Cn ti S ^^ c* ^3 ri^ SS 4 H a o M *M ^ 3 E O w o ^^^^PQ^I^QCb^JK^I^ K^K^^^^^^S^ BH^fS^QHtH^-^ .2 1 01 o E S S3 8 o CO 02 1 cd r^J o H 3 ^> 4) r2 g T3 . > 01 f* O OJ O o o 13 ^ o Co P- i^ co g g a> p *< g .p -g ^ . i o O ^P 'o'og ^^feQo> ^oSrt fe f -' r Mii Jis3|1lllMpts!t|| !.|||:s rt^ M^St^'^'^ OS'S.* ^ 0,2 s ^ '"^ 2 s ^ ctf c^ ^ rt "Ti fl "r3 P ^^Wpn^pHE-iEHQc/JCC^i-s^^-!-^^^^^ W^SHMf^Wo 1917] POSSIBLE SYMBIOSIS BETWEEN LEGUME BACTERIA AND NON-LEGUMES 135 (sapads I I I I I I I I++I I I I I 1 + (sapads XTS) wmjo/uj I I I I I I I I I I I I I I I l+l + moiooD-opndsd wimqog I I I II I I I I I wnaiivs PUB 9suMj,v wrasjj I l+l (sapads vunonjy I I I l+l I M I I I (sapads aaiqi) I I I .1+1 I (sapads Jnoj) I I I I I snuidn/2 I I I I I I l+l I I +1 PUB II I I I I I I I I I I I I ++ 1 I I I I I I I + 1 sniio.fij.vi PUB SU90S9UVO oissvg SU90S9UV9 I I (sapads uaAas) movoy uopB{ii3oni jo aoinog Botani name e g - - g rf- it! o-5 s rg t J l - tniif -t i sat ii ni ill a -i a O h O 5 fl ^ * O r*t M ^g ! g ^js 4S 4 J PM e S " o.S o t>c 2 2 II ctf -4-j ^^ ~: :;..., i . iHIUHlBfc PLATE XIII Fig. 1. Young infected cells of a nodule of hairy vetch (Vicia villosa) (same as Fig. 2, Plate XII). Stained with Flemming's triple stain. The bacteria do not show distinctly X 430 Fig. 2. Bacteroidal cells of red clover (Trifolium pratense) in a well ad- vanced but growing nodule. Cells have become vacuolated and filled with bac- teroids. Flemming's triple stain with amyl dehydration X 1080 PLATE XIV "Root nodules of Ceanothus americanus 1917] POSSIBLE SYMBIOSIS BETWEEN LEGUME BACTERIA AND NON-LEGUMES 143 the middle area. The fibro- vascular system is immediately outside this, inclosed in the cortex layer of cells. At the tip is a well defined meris- tcm made up of small, rapidly dividing cells, containing large nuclei. Few bacteria or bacteroids are found in the meristem or in the cortex ; they are confined mostly to the bacteroidal cells. Aside from possess- ing bacteroidal tissue, the nodule differs from the lateral root in that it has no central cylinder. Further, it has no root-cap and no epider- mis, but is inclosed by a protective band of corky cells. Fig. 2, Plate XI, shows a cross-section of a nodule of Trifolium pratense at about the same stage of development as that shown in Fig. 1. This section was stained with Flemming's triple stain and then mounted in dilute iodine solution in order to show the location and distribution of starch. The fibro-vascular bundles may be seen espe- cially well. The bacteroidal cells of younger nodules are strikingly different from those of older ones. In the young nodule the cells are full and closely packed. Plate XII shows sections of a young nodule of Vicia villosa. Fig. 1 of this plate shows the meristem, and Fig. 2 a section some distance back. The latter figure should be compared with Fig. 2 of Plate XI. These young infected cells are shown more highly magnified ( X 430) in Plate XIII, Fig. 1. The cells are filled with cytoplasm in which are dispersed myriads of bacteria. These bacteria occur chiefly as swarmers or as small vacuolated rods, so that it is diffi- cult to resolve them in the cytoplasm. The nuclei are quite prominent. As the nodule becomes older, the bacteria are more in evidence, the bacteroids becoming especially large and numerous. The nucleus becomes distorted and is pushed to one side of the cell, tho sometimes it disintegrates and disappears entirely, giving way to a large central vacuole, which is inclosed by a band containing mostly bacteroids. Fig. 2, Plate XIII, shows a few cells ( X 1080) in which this has taken place. (This figure shows a few cells of Fig. 1, Plate XI, more highly magnified. ) It is in this stage of development that the large, branched bacteroids, such as those shown in Plate III, are found. The fibro-vascular system extends from the meristem region to the base of the nodule, where the elements unite and communicate with the central cylinder of the lateral root. As the nodule becomes older, the bacteria further devastate the cells and probably automat- ically shut off the food supply from the root, whereupon the nodule decays and sloughs off. The so-called infection threads (F'dden, of Tschirsch; Infektion- schlauche, of Prazmowski) so frequently found, especially in young nodules, were the objects of much study. Contrary to the opinions of Dawson 23 and Peirce, 25 the infection threads are not zoogloeal strands made up of small bacilli, but are solid hyphae-like structures bearing a remarkable resemblance at times to tubes, which in fact some earlier 144 BULLETIN No. 202 [July, investigators believed them to be (see Plate XII, Fig. 3). No septae were found. The threads were more frequently seen in the meristem or in the cortex cells near the apex of the nodule. The longest one observed in a single section traversed six consecutive cells. Shorter threads were frequently encountered in the bacteroidal tissue. Fre- quently the threads were found branched, and invariably they were growing directly toward the cell nucleus or sending a branch to it. In passing thru the cell walls the thread becomes peculiarly thickened or flattened, producing a funnel-like appearance. This also occurs when the thread approaches the nucleus. With the view that the infection threads are not zoogloeal strands composed of separate bacilli, they become more difficult to explain. A possibility is that they are due to unusually stimulated bacteroids or to a number of bacteroids which fail to divide but remain attached with the resorption of the cell wall between. However, this is pure spec- ulation. 1917] POSSIBLE SYMBIOSIS BETWEEN LEGUME BACTERIA AND NON-LEGUMES 145 Part IV. NON-LEGUMES SAID TO BE CONCERNED IN THE FIXATION OF ATMOSPHERIC NITROGEN HISTORICAL The demonstration by Hellriegel and Wilf arth 5 10 of the fixation of nitrogen by legume plants and the isolation by Beyerinck of the organism from the legume nodules, stimulated interest in the root nodules found upon non-legume plants. Of the groups of non-legumes which possess these structures, those which have received the most attention are Ceanothus, Elaeagnus, Alnus, Podocarpus, Cycas, and Myrica. The earlier investigators 8 for the most part held that nodules were of fungous origin. Arzberger 49 held that the causal agents (Frankia ceanotlii and Frankia subtilis) in CeanotJius and Elaeagnus were quite similar, but that that (Frankia bruncJiorstii) of Mryica was quite dif- ferent, being of the nature of an Actinomyces. Previous to Arzberger, Hiltner 45 in 1896 claimed a fixation of nitrogen by Alnus and Elaeagnus. In 1899, Nobbe and Hiltner 46 claimed the same for Podocarpus, the nodules of which were said to be due to an endotrophic mycorrhiza. Bottomley 48 b claims to have demonstrated in 1907 the presence of nitrogen-fixing bacteria in the nodules of Cycas. In 1912 the same writer 51 reported the isolation of an organism identical with Ps. radici- cola from Myrica gale and claimed fixation of nitrogen by young Myrica plants. In the same year Spratt, 52 working in Bottomley 's laboratory, reported a similar isolation from Alnus and Elaeagnus and also from Podocarpus. 53 More recently Bottomley 54 has reported the isolation of Ps. radici- cola from CeanotJius, and shown the fixation of nitrogen in culture solutions by the organism isolated. CEANOTHUS AMERICANUS Attempts to Isolate tlie Causal Organism. As CeanotJius ameri- canus grows close at hand, material for study was easily obtained. Efforts to isolate a causal organism were persistent, covering a period from early spring to late fall. Nodules in all stages were plated, spe- cial effort being made upon the extremely young ones. With ash agar alone ninety plates were poured in duplicate. Legume nodules were frequently plated as checks upon the method, the same procedure being used in each case. Legume nodules nearly always gave good plates ; Ceanofhus nodules failed always. Variation in the seeding of the For a review of the subject Arzberger should be consulted. "The paper written in 1907 to which Bottomley refers was not found. 146 BULLETIN No. 202 [July, plates was tried. Sometimes the entire nodule was crushed with sev- eral cubic centimeters of sterile water and poured with the plate ; some- times several loops of infusion were used ; and sometimes the nodules were cut open and the tissue scraped out for plating. Ash-agar plates were inoculated direct with nodule tissue and with crushed infusion. Other media were tried. The list included Fred's agar (No. 201), Ashby's (No. 202), Spratt's (No. 203), beef -broth agar (No. 205), Conn's asparaginate agar (No. 204), Ceanothus-extract agar (simi- lar to No. 206), a mixture of Ceanothus- extract agar and ash agar, potato agar, oatmeal agar, cornmeal agar, Loeffler's blood- serum agar, and Koch 's blood-serum agar. Many plates were poured and many direct slants tried. Liquid media were not extensively em- ployed as this means of isolation is objectionable. However, Spratt's medium (No. 103) and beef broth (No. 105) were tried. In no case did a typical plate resembling those obtained in plating legume nodules result. For the most part the plates were blank except for an occasional mold or yeast. Bacterial colonies sometimes grew, but never did a single organism persist that upon examination in any way resembled Ps. radicicola. Almost invariably slants made direct from nodule tissue or crushed infusions failed to show growth. As with the plates, there was noth- ing to suggest a causal agent, either like or unlike Ps. radicicola. Little reliance was placed on the liquid cultures ; most of them showed no growth. Nitrogen-Fixation by Ceanothus americanus. In order to test the fixation of nitrogen by Ceanofhus, thirteen young plants were washed clean and planted in clean quartz sand to which lime had been added. Seven of the plants were given a nutrient solution without nitrogen, and inoculated abundantly with an infusion of crushed nodules. Six were given the same solution plus nitrogen, but were not inoculated. None of the plants fully recovered or made very vig- orous growth. After ten months all those not receiving nitrogen were dead. Two of those receiving nitrogen still survived but were not doing well. All the plants produced nodules. Seeds of Ceanothus americanus were obtained in the fall of 1915. By immersing them in commercial sulfuric acid for ten minutes a germination of about five percent was obtained. Three series of six pots were then filled with white quartz sand and four seedlings planted in each pot. Nutrient solutions were added as shown in Table 10. The plants in Series III were inoculated with an infusion of crushed Ccanottius nodule. After seven months, Series II and III had made a very weak growth ; there Avas no choice between them. Series I also had not made a very vigorous growth, but the plants were noticeably larger and greener than those of the other series. A further observation of im- PLATE XV Fig. 1. Longitudinal section of a Ceanothus amcriccmus nodule, showing parasitized zone. Stained with Flemming's triple stain and mounted in iodine to show the starch X 100 Fig. 2. Cross-section thru a similar nodule of Ceanothus americanus, showing central cylinder, some parasitized cells, starch, etc. X 100 X CD 03 O ^ s 1917} POSSIBLE SYMBIOSIS BETWEEN LEGUME BACTERIA AND NON-LEGUMES 147 portance was that Series III had no nodules, in spite of the fact that the young seedlings had been abundantly inoculated with an infusion cf crushed nodules. TABLE 10. EXPERIMENT IN NITROGEN-FIXATION WITH SEEDLINGS OF CEANOTHUS AMERICANUS Series No. of plants Inoculation Treatment Nodules I II III 24 24 24 None None Infusion of crushed Cean- otTius nodule Full nutrient solution Nutrient solution without nitrogen Nutrient solution without nitrogen None None None While the evidence submitted is not conclusive, it at least throws doubt upon the ability of CeanotTius to fix atmospheric nitrogen. Ap- parently either quartz sand is not favorable or the solutions used were not best suited to this plant. It must be considered, however, that Ceanotlius is a slow-growing shrub, and hence the demonstration would not be so easy as with quick-growing legumes. Histology of the Nodules of Ceanotlius americanus. An exten- sive description of the nodules of Ceanotlius americanus is not intended here, but it is desired to show enough of the structure so that a fail- comparison with legume nodules may be made. For further informa- tion Bottomley, Spratt, and especially Arzberger, should be consulted. In Plate XIV are shown some CeanotTius nodules. When extremely young, they are white or nearly so, and are round or slightly oval. They grow mostly along the longer axis, and become distinctly club- shaped, lacking entirely the plumpness of legume nodules. Branching commonly occurs ; one nodule divides to form two or three, and later these branches divide, the whole ultimately forming a cluster of con- siderable size. The structures are perennial, making new growth and sending out new branches each year. In appearance, as well as to the section knife, they are quite woody, a point that further distinguishes them from legume nodules. The parasitized zone of a Ceanotlius nodule may be seen in Plate XV, Fig. 1. The section was mounted in iodine solution, as before described, to show the starch. The meristem and central cyl- inder do not show. Fig. 2 of the same plate shows a cross-section thru a. similar nodule. The central cylinder is well developed and possesses a well defined endodermis. In the nodule cortex surrounding the en- dodermis, there are first several layers of small, apparently vacant cells, and then a zone of parasitized cells, which are rather loosely scattered in this area. The accumulation of starch in the cells sur- rounding the parasitized zone shows clearly. As in the legume nodule, there is no epidermis and no root-cap, but there is a protective layer of corky cells. 148 BULLETIN No. 202 [July, Plate XVI shows some of the parasitized cells. Fig. 1 is magnified 430 diameters, and Figs. 2 and 3, 1,080 diameters. The dark bodies are said by Arzberger to be sporangia of a fungus, the hyphae of which may be seen within the cells at certain stages. He designates the fnngus as Frankia ceanofhi Atkinson. While not agreeing with Arz- berger in all the details, the writers accept the fungous 1 conceptions as the true ones. The parasitized cells are tough and horny in consis- tency. When nodules were crushed for plating, these cells remained intact and were frequently seen distributed thruout the agar plates, where they were at first mistaken for colonies. No growth was pro- duced by them, however. These parasitized cells clearly bore no resemblance to the bacteroidal cells of the legumes. Summarizing, the nodules of Ceanofhus are unlike those of the Leguminosae in the following points : 1. The CeanotJius nodules differ in external appearance from legume nodules ; also, they are quite woody. 2. They are perennial structures, making new growth and pro- ducing new branches each year. The mode of branching is different from that of legume nodules. 3. They contain a well developed central cylinder, resembling in this respect a lateral root, of which they may be considered as a modification. 4. The parasitized cells are not closely packed as in the case of legume nodules, the characteristic bacteroids of legume nodules are not present, and the cells do not develop a central vacuole as do bacteroidal cells. Instead, the parasitized cells bear every indication of containing fungous hyphae. 5. No infection threads were found in the nodules of CeanotJius. CYCAS REVOLUTA Attempts to Isolate the Causal Organism. Repeated attempts were made to isolate the causal organism from nodules of Cycas revo- luta obtained from a greenhouse plant. The results were not wholly without success. The nodules of Cycas differ from those of the other five groups of non-legumes producing nodules in that the older ones become infected with a blue-green alga,* undoubtedly a secondary infection, which renders the nodule less solid and compact. In a cross- section cut from one of these older nodules the algal zone can easily be seen with the unaided eye. From several algal-infected nodules three forms of bacteria wer-e isolated, none of which resembled Ps. radicicola. Two were small, deeply staining rods, and the third was a larger rod. These three organisms were tried in sand pot cultures upon Pisum arvense, Vicia villosa, Trifolium pratense, Medicago sativa, Melilotus alba, Pliaseolus "See Spratt w M ; also Life. 47 1917] POSSIBLE SYMBIOSIS BETWEEN LEGUME BACTERIA AND NON-LEGUMES 149 vulgaris, Vigna sinensis, Qlycine hispida, Lupinus perennis, Arachis liypogoea, Trigonella foenum-graecum, Desmodium canescens, Amphi- carpa monoica, Ornithopus sativus, and Onobrychis sativa. Only one plant produced nodules Trigonella, on which three appeared. This fact, however, is not regarded as significant, since Melilotus and Medicago were without nodules. ( The nodules of Trigonella, Melilotus, and Medicago, it has been shown, are produced by the same organism.) Nodules due to chance inoculation are to be expected when open pots are used. It was further observed that the roots of most of the plants were distinctly brown and unhealthy, as tho attacked by a brown rot. The roots of Pisum, Vicia, PJiaseolus, and Lupinus seemed especially to be injured. A small piece of an unhealthy lupine root was teased apart and examined, disclosing a host of motile bacteria. Young Cycas nodules in which the algal zone was not present were plated, but without success. This seemed to indicate that the organ- isms first isolated were not causal agents, but that they followed the alga. Examination of the algal infected nodules disclosed a very loose, open structure; indeed, the whole nodule lacks the compactness of a Ceanothus nodule. The chance for entrance by the alga and later by foreign bacteria is very great. It would be surprising indeed if bac- teria were not found in these older nodules. ALNUS, ELAEAGNUS, AND MYRICA Spratt, 52 after pointing to the demonstration by Hiltner 45 of the fixation of nitrogen by Alnus and Elaeagnus, reported the isolation of Ps. radicicola from the nodules of these plants: The results reported in connection with the experiments are subject to the following criticisms : First : The demonstration by Hiltner of the fixation of nitrogen by Alnus and Elaeagnus is not nearly so convincing as Hellreigel 's and Wilfarth's 10 discovery of the symbiosis between Ps. radicicola and legumes. Second: Spratt 's method of isolation is at fault. Spratt steri- lized nodules and dropped them into flasks containing a liquid medium, incubating them two days. Obviously a single foreign organism in two days becomes a multitude, and no doubt a pure culture. It is not clear whether her agar plates were made from fresh nodule infusions or incubated material as described above, tho it appears that the latter was the case. The method is unsafe unless carried out on a very extensive scale (and then it is questionable), but Spratt used but one culture flask for Alnus and one for Elaeagnus, leaving one check, which may as well have been omitted. (Agar plates poured from legume nodules as before described, seldom fail to give good plates in this laboratory. If Ps. radicicola is present in Alnus and Elaeagnus, this method should easily demonstrate it.) 150 BULLETIN No. 202 [July, Third: The Kiskalt amyl-gram stain described by Harrison and Barlow and used by Spratt does not identify Ps. radicicola. Numerous gram-negative soil organisms lose the stain (aniline- gentian- violet) in ethyl alcohol, but retain it when amyl alcohol is used. Fourth: The coccoid form described by Spratt is not analogous to the bacteroids of Ps. radicicola. There is no evidence to show that Ps. radicicola ever assumes the shape or characteristics described by Spratt. The writers have never observed it. The only form of Ps. radicicola approaching a coccus form is the extremely small, oval schwarmer of Beyerinck. The form described by Spratt is entirely too large for the schwarmer. Fifth : The fixation* of nitrogen in culture solutions is neither a test for Ps. radicicola nor a proof of symbiosis. With the legume organism, the fixation of nitrogen in culture solutions is not significant when compared with fixation in the nodule. Besides, many soil organ- isms have been attributed this power of fixation in nutrient solutions. Attempts to Isolate the Causal Organism. Nodules from Alnus glutinosa and Myrica gale were obtained and plated out. The results were negative. However, the trials were not extensive because the available material was limited. Ash agar, Spratt 's agar, and beef- broth agar were used as media. CONCLUSIONS From the foregoing discussion, the following conclusions are drawn: 1. The root-nodules of Ceanoihus, Cycas, Alnus, and Myrica are not caused by Ps. radicicola. 2. It is conceivable that Ps. radicicola might enter the nodules of Cycas as a secondary infection and function symbiotically, but its presence was not demonstrated. 3. The evidence that Elaeagnus and Podocarpus nodules are caused by Ps. radicicola is not conclusive. 4. Proof that these six groups of plants are concerned with the fixation of atmospheric nitrogen is wanting. 1917] POSSIBLE SYMBIOSIS BETWEEN LEGUME BACTERIA AND NON-LEGUMES 151 Part V. ATTEMPTS TO DEVELOP A SYMBIOSIS BETWEEN LEGUME BACTERIA AND NON-LEGUME PLANTS Previous Attempts. In 1893, Schneider, 18 at the Illinois Experi- ment Station, under the direction of the senior author, cultivated nodule bacteria from Pliaseolus vulgaris upon bean-extract agar, then upon a mixture of bean-extract and corn-extract agar, and finally upon pure corn-root-extract agar. Transfers were made every sixth day. After the cultures had grown for a month upon the pure corn-root extract, they were applied upon germinating seeds of corn and oats. Tho the inoculated corn plants produced no nodules, Schneider claimed that they were more thrifty than the uninoculated plants. He de- scribed and figured the infection of some of the root-hair cells, as well as some of the epidermal and parenchymal cells. No effect was noted upon oats. That the senior author was intensely interested in this problem of developing a symbiosis between legume bacteria and non-legume plants is shown by the fact that when the opportunity presented itself after over twenty years, he took up the problem where Schneider* had left it. Other attempts in this direction have been reported. Stutzer, Burri, and Maul 19 inoculated mustard plants with nodule bacteria which had gradually become accustomed to a mustard-plant medium, but without success. Grosbiisch 31 experimented with Graminae, but his results were negative. Lemmermann 27 studied the difference in nutrition between the Lcguminosae and the Graminae. He believed that the reasons for the existence of bacterial symbiosis in the Leguminosae and not in the Graminae are, namely, the smaller transpiration current, the higher acidity of the root sap, and the greater root development of the former as compared with the latter. Preliminary Discussion. How long ago symbiosis between the Leguminosae and the nodule bacteria began cannot be estimated. The conditions under which the first infection took place are but a matter of conjecture. Only a mass of contradictory literature concerning this symbiosis existed as a basis in attempting to develop a symbiosis be- tween these bacteria and non-legume plants. There seemed, however, The following excerpts are quoted from a footnote by the senior author introducing Schneider 's work in 1893. "Can the organisms be made to grow upon these roots (grasses or cereals) by artificial means? . ' ' It must be confessed that it would have been exceedingly hazardous for any one to have expressed an affirmative opinion upon this question; but the vast importance of the matter made it desirable to try anything which gave the least promise of success While little direct evidence has been gained in favor of ultimate success, it is desirable to publish an account of the work so far done, with the hope of being able at some future time to add greatly to the infor- mation now obtained. ' ' 152 BULLETIN No. 202 [July, to be several points of attack which offered possibilities. There was some hope that the organism might be modified or changed. By accustbming.it to media containing juices of non-legume plants, it was thought that it might become so modified that it would infect such plants. Injury to the plant, especially nitrogen starvation, was a possibility. Mechanical injury, however, seemed useless, since if it would develop a symbiosis the cultivation of crops would long since have accomplished it. The non-legume plants bearing nodules, such as CcanotJius, Elaeagnus, and Cycas, offered another possibility. It was hoped that if Ps. radicicola was present in the nodules, the organism would be more adaptable to other non-legume plants. In addition, the fact that the symbiosis appeared not to> be confined to the Leguminosae alone was a great encouragement. It became evident, however, on investigating nodules of non-legumes that Ps. radicicola was not the causal organism; and furthermore, it seemed very doubtful if these plants were concerned with fixation of atmospheric nitrogen. Another plan was to obtain a non-legume plant standing in close botanical relationship to the legumes, and attempt to inoculate it with legume bacteria. The organism from cowpea nodules offered the greatest possibilities, since it seemed less particular in its selection of a host plant. Moore, 28 in 1905, reported that by inoculating legumes with nodule bacteria from Pisum sativum which had been grown for two weeks upon nitrogen-free media, he was able to produce nodules upon many genera. He stated that this was but a single demonstration of numer- ous successful cross-inoculations. It appears from his work that it was necessary only to grow the organisms upon nitrogen-free media in order to break the special adaptations. Nobbe and Hiltner, 24 in 1900, claimed that they were able to make the nodule bacteria from peas produce nodules upon the roots of beans, and vice versa. Laurent, 14 Maze, 21 and Kellerman, 37 among others, have reported similar successful cross-inoculations. EXPERIMENT I: COMPARISON OF NITROGEN AND NITROGEN-FREE MEDIA FOR THE GROWTH OF PS. RADICICOLA In order to compare nitrogen and nitrogeii-fvec media for the growth of Ps. radicicola, bacteria from Melilotus alba and Trifolium pratense were transferred to Freudenreich flasks containing standard beef -broth agar (No. 205) and Fred's synthetic agar (No. 201). The cultures were kept in the incubator at room temperature for thirty months without transfer. Duplicate cultures were transferred to test-tube slants once a month. At the" end of the thirty months all cultures were transferred to ash-agar slants for comparison. All 1917} POSSIBLE SYMBIOSIS BETWEEN LEGUME BACTERIA AND NON-LEGUMES 153 were alive and capable of producing nodules upon plants in test-tube cultures after Garman's method. Furthermore, it is important to note that the cultures retained their special adaptation to the original liost plant and their cultural individualities as described on pages 136 and 139. EXPERIMENT II: COMPARISON OF ASH AGAR AND BEEF-BROTH AGAR FOR THE GROWTH OF PS. RADICICOLA Ash agar and beef-broth agar were compared as in Experiment I. Cultures of Trifolium pratense, Melilotus alba, Vigna sinensis, Gly- cine Tiispida, Robinia pseudo-acacia, and Arachis hypogoea were transferred to Freudenreich flasks containing ash agar and beef -broth agar. Duplicate test-tube cultures transferred once a month were kept. The culture of Robinia pseudo-acacia upon beef -broth agar was lost because of a mold. After seventeen months the cultures were transferred to ash-agar slants for comparison. All were alive (except the Robinia) and all retained their individual habit of growth. Trifolium pratense and Melilotus alba were tested by Garman's method and the others in sand pot cultures. (The remaining Robinia cul- tures were not tested except for growth.) All those tested were capable of producing nodules upon their original hosts. A few cross- inoculations were tried but failed. No difference in virulence was noted. EXPERIMENT III: GROWTH OF PS. RADICICOLA ON TOMATO-STEM SLANTS For the purpose of infecting tomato plants, cultures of Melilotus alba and Trifolium pratense were grown upon tomato-stem slants (No. 421) placed in tubes containing standard beef broth. Transfers were made once a month. After several months, distilled water was substi- tuted for the broth. At the end of twenty-three months the cultures were transferred to ash-agar slants. Eight cultures of Melilotus alba and two of Trifolium pratense were examined. All grew readily when transferred to ash-agar slants. Tested in agar-tube cultures, all pro- duced nodules upon their original hosts. Melilotus alba bacteria, how- ever, failed to inoculate Trifolium pratense plants, and Trifolium bacteria failed with Melilotus seedlings. EXPERIMENT IV: COMPARISON OF ASH AGAR AND CONN'S ASPARAGINATE AGAR AND THE EFFECT OF SUNLIGHT ON GROWTH AND VIRULENCE OF PS. RADICICOLA This experiment was designed not only to compare nitrogenous and non-nitrogenous media, but also to note the effect upon growth and virulence of exposure to direct sunlight. 'Cultures of organisms from various legumes were transferred to Freudenreich flasks of 25-cc. 154 BULLETIN No. 202 [Julii, capacity, two sets containing Conn's asparaginate agar and two ash agar. All cultures were incubated for three days at room tempera- ture, after which one set of the asparaginate-agar flasks and one of the ash-agar were removed to the greenhouse and left exposed to the sun- light. The slopes were turned toward the south to give maximum exposure. The checks, fewer in number, were left in the incubator. The experiment covered three months from March 2 to June 2, 1916. The temperature in the greenhouse varied from 18 to 42.5 C. The highest temperature recorded within a similarly prepared flask was 47 C. (thermometer shaded). In Table 11 is shown the arrangement of the experiment. TABLE 11. ARRANGEMENT OF CULTURES TESTED ON ASH AGAR AND CONN 's ASPARA- GINATE AGAR IN SUNLIGHT AND IN DARKNESS : EXPERIMENT IV Source of organism In sunlight In darkness Common name Botanical name Ash agar | Conn's agar Ash agar [ Conn's agar FlasJc No. Flask No. Flask No. FlasJc No. Acacia Acacia melano- Tick trefoil xylon Desmodium canes- 6011 (died) 6026 cens 6012 6027 . Dyer's green- weed Genista tinctoria 6013 6028 Soybean Sweet pea White sweet Glycine hispida Lathyrus odoratus 6014 6015 6029 6030 6016 603i clover Bean Trailing wild bean Melilotus alba Phaseolus vulgaris Strophostyles helvola 6017 (lost) 6019 6020 6032 6034 6035 6018 6033 Red clover Broad bean Cowpea Trifolium pratense Vicia faba Vigna sinensis 6021 6023 6024 (died) 6036 6038 6039 (died) 6022 6025 6037 6040 At the end of three months all cultures were transferred to ash- agar slants. Flask No. 6017 had been broken; the cultures in Nos. 6011, 6024, and 6039 had died. Of the surviving cultures those which had been kept in darkness recovered the most quickly. It was also noted that the organisms grown upon Conn's asparaginate agar were the most vigorous. The ash-agar cultures which had been exposed to sunlight were the slowest in recovery. After several transfers upon ash agar, the cultures resumed their normal appearance. They were then tested out for virulence (except Phaseolus and Stroplwstyles) , and it was found that the ability to produce nodules had not been affected. The cultures of Melilotus and Trifolium were tested by Gar- man's method for ability to cross-inoculate seedlings of Trifolium and Melilotus respectively, but the cultures were virulent only upon the original host. The spring was cool and cloudy for the most part, tho there were some clear, hot days. 1917] POSSIBLE SYMBIOSIS SETWEEN LEGUME SACTERIA AND NON-LEGUMES iS5 EXPERIMENTS ATTEMPTING THE INFECTION OF NON-LEGUME PLANTS WITH PS. RADICICOLA The following experiments attempting the infection of non-legume plants with Ps. radicicola were but preliminary. In examining inocu- lated plants, attention was given only to any unexplained vigor and to the presence or absence of abnormal root conditions. Histological technic was not employed. EXPERIMENT V: ATTEMPTED INFECTION OF TOMATO SEEDLINGS WITH SWEET-CLOVER BACTERIA Very young tomato seedlings were transferred from flats of soil to one-gallon pots of limed white quartz sand. There were in all one hundred and fifty plants. Half were given a full nutrient solution,* and half were given a similar solution but without the nitrogen. Copious in- oculations were made frequently with bacteria from sweet clover which had been grown for three weeks, with frequent transfers, upon a decoc- tion of whole tomato plants plus two percent cane sugar and one per- cent peptone (Medium No. 111). After one month the plants were carefully washed free from sand and examined. Those which had been receiving nitrogen were decidedly more thrifty than the others. No abnormal conditions were observed in the roots. EXPERIMENT VI: ATTEMPTED INFECTION OF TOMATO SEEDLINGS WITH SWEET-CLOVER BACTERIA IN THE PRESENCE OF COPPER SULFATE Tomato seedlings which had been grown in flats of soil were transferred to paper boxes containing limed white quartz sand, one plant to each box. These boxes (2" x 2" x 4%") were arranged in a wooden frame, sixteen rows of sixteen each, making two hundred and fifty-six in all. Nutrient solutions made up as before were used, except that the nitrogen was varied as indicated in Table 12. Inoculations were made at the time of transplanting and again after two weeks with TABLE 12. TREATMENT APPLIED TO TOMATO SEEDLINGS: EXPERIMENT VI Section No. of No. (plants Nitrogen treatment Copper-sulfate treatment 738 32 Full nutrient solution* (10 cc. stock solution per liter water) None 739 32 Full nutrient solution 50 cc. of 1:2500 solution 740 32 Full nutrient solution 50 cc. of 1:1000 solution 741 32 Full nutrient solution 50 cc. of 1:500 solution 742 32 Nutrient solution without nitrogen None 743 32 Double nutrient solution None 744 32 Nutrient solution without nitrogen 50 cc. of 1:1000 solution 745 32 Double nutrient solution 50 cc. of 1:1000 solution "See footnote, page 125. 156 BULLETIN No. 202 [July, bacteria from sweet clover which had grown upon tomato-infusion peptone (No. 111). After ten days copper sulfate was applied to the sections indicated in amounts intended to stimulate growth, to just hinder growth, and to seriously retard growth. The treatment is shown in Table 12. The plants were examined after four weeks. Those which had received the normal amount of nitrogen showed the best development. The 1 : 2500 solution of copper sulfate stimulated both root and top development ; the 1 : 1000 solution was slightly injurious ; and the 1 : 500 damaged the plants seriously. No abnormal conditions of the roots were observed, except where the 1 : 500 copper-sulfate solution was applied, in Avhich cases the injury was apparent. EXPERIMENT VII: ATTEMPTED INFECTION OF TOMATO SEEDLINGS WITH SWEET-CLOVER BACTERIA IN SOIL AND IN SAND WITH VARIED NITROGEN TREATMENT Tomato seedlings growing in sand and in soil in an arrangement similar to that of Experiment VI were inoculated with bacteria from sweet clover which had been grown for ten months upon tomato-stem slants (No. 421) . Bacteria were applied at the beginning of the experi- ment and at intervals of a week thereafter. The nitrogen treatments used were varied as shown in Table 13. TABLE 13. TREATMENT APPLIED TO TOMATO SEEDLINGS IN SOIL AND IN SAND: EXPERIMENT VII Section Sand or 'soil No. of plants Treatment Eemarks A Sand 256 Full nutrient solution B Sand 256 Nutrient solution without A small amount of nitrogen nitrogen was added later, as the plants were starving. With 4 the exception of a few very weak plants, how- ever, all died C Sand 224 Harrison-Barlow wood-ash (No. 100) D Soil 288 Tap water Plants grew very vigor- ously and were cut back. 160 plants were given 1:500 copper sulfate solu- tion to further check the growth E Sand 128 Nutrient solution with J io nitrogen F Sand 112 Nutrient solution with ni- trogen trebled G Soil 112 Tap water Plants were cut back H Soil 112 Nutrient solution with ni- trogen trebled i Plants were cut back 1917] POSSIBLE SYMBIOSIS BETWEEN LEGUME BACTERIA AND NON-LEGUMES 157 In general it may be said that the plants in soil were the. most .vigorous. Those in sand without nitrogen made very weak growth or died. Those watered with ash solution also made very little growth, probably because of a lack of nitrogen. The roots of the plants bore no abnormal structures. EXPERIMENT VIII: ATTEMPTED INFECTION OF COMMON MORNING GLORY 'WITH SWEET-CLOVER BACTERIA The plan was much like that of Experiment VII, except that com- mon morning glory (Convolvulus major} was used. Seeds were planted in sand and in soil, using the paper boxes before described. There were 1,556 plants in all. Inoculations were made at the time of planting and again in two weeks with nodule bacteria of sweet clover which had been grown for one month, with frequent transfers, in an infusion of morning-glory plants plus two percent cane sugar and one percent peptone. Unfortunately some of the records were lost, but Plate XVII gives a general idea of the experiment. The inoculation produced no visible effect. The roots were carefully exam- ined but showed no unusual conditions. EXPERIMENT IX: ATTEMPTED INFECTION OF TOMATO SEEDLINGS WITH SWEET-CLOVER BACTERIA AND WITH A COMPOSITE INFUSION OF MANY LEGUME BACTERIA The experiment involved 1,268 tomato seedlings grown in paper boxes filled with limed white quartz sand. These were divided into two equal sections. Those in one section were given the full nutrient solution, while those in the other were given the nutrient solution without nitrogen. Half the plants in each section were inoculated with sweet-clover bacteria which had been grown for twelve months upon tomato-stem slants (No. 421). The other half were inoculated with a composite of all the cultures of nodule bacteria on hand. There were cultures from forty-five plant species, including twenty different generic groups. Some were recent isolations, but most of them had been kept as stock cultures for one to two years. All had been grown upon ash-agar slants. The inoculations were in every case without' apparent effect. EXPERIMENT X: ATTEMPTED INFECTION OF STRAWBERRY PLANTS WITH SWEET-CLOVER BACTERIA AND WITH A COMPOSITE INFUSION OF BACTERIA FROM SEVEN SPECIES OF ACACIA Young strawberry plants, one hundred and twenty in all, were planted in one-gallon pots of sand and of soil and treated as shown in Table 14. Half the plants in each series were inoculated with sweet- 158 BULLETIN No. 202 [July, PLATE XVII Experiment VIII: Morning-glory plants grown in sand and in soil inocu- lated with sweet-clover bacteria grown for one month in morning-glory infusion media 1&17] POSSIBLE SYMBIOSIS BETWEEN LEGUME BACTERIA AND NON-LEGUMES 159 TABLE 14. TREATMENT APPLIED TO STRAWBERRY PLANTS IN SAND AND IN SOIL: EXPERIMENT X Series No. of plants Medium Treatment A B C D 30 30 30 30 Good potting soil 1 part soil to 4 parts sand Washed yellow sand Washed yellow sand Tap water only Tap water only Full nutrient solution Full nutrient solution without nitrogen clover bacteria; the other half were inoculated with a composite in- fusion of bacteria from seven species of Acacia^ Heavy inoculations were made frequently ; the bacteria used had been grown upon ash-agar slants. The plants did well at first, but later became infested with red spiders and in spite of sprays did not make a satisfactory growth. The results were negative. 160 BULLETIN fro. 20% [July, SUMMARY 1. The nodule bacteria studied were found to be true Sctiizo- mycetes, actively motile by means of a single polar flagellum. 2. These bacteria may be divided into groups according to the host plants to which they become specifically adapted. In addition to the cross-inoculations previously known, many new ones were found to exist. These are given under Group III, page 136. 3. In addition to these special adaptations, there are among the various nodule bacteria serological and cultural differences which are permanent, giving perhaps a legitimate basis for the belief that distinct species exist. In numerous other characteristics, however, the nodule bacteria are so strikingly alike, and as a whole they differ so widely from any other species of bacteria, that it seems more consistent to regard the adapted forms as varieties of the single species Pseudomo- nas radicicola. 4. The legume nodule originates in the root-cortex, much as does the lateral root, but here the similarity ends. The nodule consists chiefly of a mass of parenchymal cells which are devastated by the nodule bacteria giving way to the bacteroid forms of the invading organism, which then make up the greater part of the cell contents. 5. The nodules of the non-legumes Ceanothus, Cycas, Alnus, and Myrica, said to be concerned with the fixation of atmospheric nitrogen, are not caused by Pseudomonas radicicola. The nodules of Ceanothus are wholly different morphologically from those of the Leguminosae. The evidence that the nodules of Elaeagnus and Podocarpus are caused by these organisms is not conclusive. Furthermore, the proof that any of these six groups of plants are concerned in the fixation of atmospheric nitrogen is not conclusive. 6. The adaptations of the nodule bacteria are constant. Such factors as the use of organic or inorganic substances in the medium, the acidity or alkalinity of the medium, and the presence or absence of combined nitrogen in the same, do not affect the virulence nor break the special adaptations. The virulence and specificity are bound up with the life of the organism. 7. The preliminary experiments here reported attempting the infection of non-legume plants with nodule bacteria failed. 8. No conclusions can be drawn as to the possibility or probability of developing or finding nodule bacteria that will grow on non-legume plants. The constancy of the special adaptations and the fact that no plants other than legumes harbor the organisms in question, as had been supposed, have been discouraging and to some degree limit the hope of ultimate success. 1017] POSSIBLE SYMBIOSIS BETWEEN LEGUME BACTERIA AND NON-LEGUMES 161 PART VI. BIBLIOGRAPHIES 1 (a) SYMBIOTIC NITROGEN FIXATION BY LEGUMES 1687 MALPIGHI. Anatomic plantarum pars sec. de gallis. Op. (1687), 2, 126, (Leiden). 1825 DECANDOLLE. Memoircs sur la famille des L6gumineuses (1825)j 22. (Paris). 1837 BOUSSINGAULT. Becherches chimiques sur la vegetation enterprises dans le but d 'examiner si les plantes prennent de 1 'azote de 1 'atmosphere. Ann. Sci. Nat. Bot. (1837), 10, 257. 1838 BOUSSINGAULT. 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(1900), 26, 57-77. DEHERAIN ET DEMOUSSY. Recherches sur la Ve'ge'tation de Lupins. Deuxieme partie: Lupins bleus (Lupinus angustifolius). Ann. Agron. (1900), 26, 169-196. HARTLEB. Die Morphologie und systematische Stellung der sogenannten Knollchenbakterien. Chem. Ztg. (1900), 24, 887-888. HILTNER. Ueber die Ursachen welche die Grosse, Zahl, Stellung, und Wirkung der Wurzelknollchen der Leguminosen bedingen. Arb. K. Gsndhtsamt., Biol. Abt. (1900), 1, Heft 2, 177. Abst. in Just's Bot. Jahresber. (1900), 28, Part 1, 47. HILTNER. Ueber die Bakteroiden der Leguminosenknollchen und ihre wirkiirliche Erzeugungausserhalb der Wirtspflanzen. Centb. f. Bakt. 2 Abt. (1900), 6,273-281. LUTOSLAWSKI. Beitrag zur Lehre von der Stickstoffernahrung der Legum- inosen. Ber. Landw. Inst. Univ. Halle. (1900), Hefte 14, 36. See also Jahresber. Agr. Chem. (1900), 43, 125-126. "NOBBE UND HILTNER. Kunstliche Ueberf iihrung der Knollchenbakterien von Erbsen in solche von Bohnerr. (Phaseolus). Centbl. f. 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(1901), 7, 897-912. 1902 BREFELD. Versuche ueber die Stickstoffaufnahme bei den Pflanzen. Centbl. f. Bakt. 2 Abt. (1902), 8, 24-25. BUHLERT. Ein weiterer Beitrag zur Frage der Artenheit der Knollchenbak- terien der Leguminosen. Centbl. f. Bakt. 2 Abt. (1902), 892-895. BUHLERT. Untersuchungen ueber die Artenheit der Knollchenbakterien der Leguminosen und Ueber die landwirtschaftliche Bedeutung dieser Frage. Fiihling's Landw. Ztg. (1902), 51, Heft. 11, 385-391, and Heft. 12, 417-427. See also Centbl. f. Bakt. (1902) 2 Abt., 9, 148-153, 226-240, 273-285. HILTNER. Ueber die Impfung der Leguminosen mit Reinkulturen. Dent. Landw. Presse (1902), 29, No. 15, 119-120. HOPKINS Alfalfa on Illinois soil. 111. Agr. Exp. Sta. Bui. 76 (1902). MOORE. Bacteria and the nitrogen problem. U. S. Dept. Agr. Yearbook (1902), 333-342. NEUMANN. Die Bakterien der Wurzelknollchen der Leguminosen. Landw. Vers. Stat. (1902), 56, 187-206. NEUMANN. 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