CROSS INOCULATION AS INFLUENCING NITROGEN FIXATION BY JAMES GEORGE KALLAS THESIS FOR THE DEGREE OF BACHELOR OF SCIENCE IN AGRICULTURE COLLEGE OF AGRICULTURE UNIVERSITY OF ILLINOIS 1922 , UNIVERSITY OF ILLINOIS JUTlg § a 19 fr fr. THIS IS TO CERTIFY THAT THE THESIS PREPARED UNDER MY SUPERVISION BY J % . . .Q.Sfi £g£ . 1 i&S. entitled £ JTQg.^. .EA&ftkUl A IS APPROVED BY ME AS FULFILLING THIS PART OF THE REQUIREMENTS FOR THE DEGREE OF £aS .fc e.l.G Jf. . .Q £ . . 5.9. i .ett« £ . . .itt . . £& X'iSU 1 .t.UX.ft HEAD OF DEPARTMENT OF Mr.Q &QM. TABLE OF CONTENTS Introduction. 1 Review of Literature 2 The Proo lexa 2 Experiment I 3 Experiment II 8 Part II 12 Review of Literature 12 Experiment III 15 SvuSi&ry IS Acknowledgment 19 Bibliography * 20 TEE INFLUENCE OF CROSS- INOCULATION ON NITHOGEN FIXATION . INTBODUCTION llany cross- inoculations are recognized among the bacteria common to various legumes. The most complete and extensive work dealing :ith the proof of cross-inoculations has resulted from the v/ork of Burrill and Hansen, and Whiting and Hansen, and whiting and others. The groups established by these workers have been verified by other workers. While the groups established contain the most important legua.es now under cultivation, it is clearly recog- nized that many other crosses yet undetermined probably occur. It is conceded that cross-inoculation results in the production cf nodules and in an increased growth of the plants, together with an increase in the nitrogen content. The fundamental relationships responsible for an or- ganism adapting itself to legu.es of widely different characteristics have not been discovered. Whether the morphology of the root structure of the legumes in a given group plays a part of this adaptation is, cf course, not known but snould be carefully considered. The chemical nature of the cell sap cf the plants within a given group _.lso offers a possible line of investigation which may assist in clearing -up what appear to be peculiar relationships of the nodui bacteria. It might be assumed that a given cheudcal substance, such as an alkaloid, existed in ail the species contained in a given group. Another con- sideration which should be carefully eliminated deals with the production cf specific enzymes by the organisms associated with the various groups. There are two distinct organises responsible for the inoculation cf legumes from the standpoint of the bacteriological classification. One is a Digitized by the Internet Archive in 2016 https://archive.org/details/crossinoculationOOkall typical peritrichic ‘bacillus and of this form there are probably numerous varieties, such as these inoculating red clover, alfalfa, garden peas, and garden beans. The other organise is a g seudononas possessing a flagellum, net, however, typically attached. The attachment of the flagellum has been described as on the corner instead of at the pole. Two totally distinct varieties of the pseudomonas are definitely recognized, that of the cowpeas and that of the soybeans. In all probability, there are others, although they have not yet been found. It was not until puite recently that pure cultures of these organisms were obtained because of a lack of suitable methods for testing out the purity of the organism. The work reported by Uniting and Hansen was conducted with cultures, the purity of which was absolutely proved before and after making the crosses reported. The investigation reported in this paper deals with the efficiency of the process cross-inoculation, from the standpoint of the i~- ediate adaptation of an organism in a given group to any -ember of that group upon which it has not recently produced nodules. A specific example would be the inoculation of cowpeas with bacteria previously isolated from, peanuts, in comparison with bacteria previously isolated fro- the co\*p sa itself. In other -words, would there be an advantage in always using for inoculation bacteria from like legumes instead of bacteria from any legumes within an established group . REVIEW 0? LITERATURE A survey of the literature failed to reveal any work dealing with the problem under study. THE PROBLEM The first part of this *rcrk was planned to measure the nitrogen content of legumes inoculated -with bacteria common to the legume to be used in the study and with other bacteria producing nodules on the same legume. In this roanner, the differences in nitrogen content in the presence cf nodule bacteria . I - 7 - recently from the legume under study and in the _resence of nodule bacteria from widely different legumes might be obtained. EXPSEII4ENT I Cross-Inoculation of Cowp eas as Influencing Fixation. Cowpeas were selected as the legume to be inoculated with peanut, par- tridge pea, beggarweed, and lima bean a ong the nodule bacteria, and in addition with Eadiobaoter and Azotobacter. The differences that night develop in the growth and nitrogen content of cowpea plants with these various sources of inoc- ulation were to be used as the criteria for judging the efficiency of cross- inoculation vs. inoculation with bacteria recently from the cowpea. Preparation of Sand . The s.-nd used was a high = rude clean white sand. It was sterilized in 1-gallon earthenware jars at 350° F. for several hours. Tests for nitrate failed to show its presence in the sand grains. After sterilization, the jars were allowed to aerate for 48 hours. Approximately 5 gra s of nitrogen- free, re-precipitated CaCQ- was added to each jar and thor- oughly mixed with a sterile spatula. The moisture content of the sand was established at 12 percent and maintained at about that percent throughout. Seeds ? lant ed . Seeds possessing unbroken seed coats were selected. In the first experiment, the cowpeas were selected from a large sample; in the second experiment, the sample of garden x sas was somewhat smaller. The seeds were sterilized in 1 to 500 mercuric chloride for 10 minutes. They were washed with distilled water several times and they were allowed to dry and were then placed in a sterile jar and kept ready to use. Plant Food Applied. The Jars were ^.reviled with all plant food with the exception of nitrogen. The plant food was prepared fro- - pure nitrogen-free chemicals and a - onia-free distilled water. Solution No, 1. Phosphorus and potassium: Dissolve 28 grams of K__P?04 - Il- ia 2p00 c. c. au-vonia-f res wat er. Use 10 o. c. per jar. Solution No. 2. Magnesite.: Dissolve 20 grams cf '-IgSQU in 2500 c. c. ammonia-free water. Use 10 c. c. per jar. Solution No. 3* Iron: Dissolve 0.1 gram of FeCl-? in 250 c. 0 . amaonia- free water. Use 1 c. o. per jar. The solutions were applied as follows: 10 c. 0 . K 9 HPO 4 -f- 10 c. c. figSO^ 4- 1 c. c. FeCl - made up to a total volume of 100 c. c. with aramonia-f water for each jar. The first application was :.-ade at the time of planting, the second, two weeks later, and the subsequent applications at intervals of one week. Source of Inoculation . The organises used for the experiments in this work were transferred cultures froi' Dr. A. L. Whiting's pure stock cultures. The organisms were & rown in standard :annite agar edia the composition of which is as follows: Agar (powdered) 15 g ra^-s Uannite 10 tf DNO- 0.2 it K^HPOji 0.2 1 ? MgS0 4 0.2 11 NaCl A A • L it CaSOh 0.1 tt FeClj 3 drops of 10fo so lution CaCOj ■ excess . Water 1000 c . c . The inoculation was applied as a suspension over the seeds at the time of planting. The seeds in Exp eri ent 1 were planted October 13 » 1921. There were 12 seeds planted in each jar. The kinds cf inoculation as well as the - 5 - ana lysis of the plants is shown in Table 1. The plants were treated for mildew three different tines with a sulfur compound which contained no nitrogen. Some of the plants were effected more than others and their growth was somewhat stunted. The plants grown in the jars inoculated with Radio barter and Aaotobacter organisms began to turn yellow' five v/eeks after planting and finally all of the plants grown in these jars turned yellow and growth ceased. The plants in the ether jars were larger and had larger leaf surface a*id continued to grow. The only difference observed between the jars inoculated with the nodule organisms was found in the two jars inoculated with peanut bacteria. The plants in these jars were more vigorous and a little larger than the ethers. This, however, night have been due to the fact that they were not attacked by ildew as badly as some of the other jars. The control jars began to turn yellow at the time the Azote one ter and Radi obac ter jars did, but they were contaminated later and began to turn green again. The plants were harvested December l4, 1321. The check jars were con- taminated, nodules were found in their roots. The jars inoculated with Azotcbacter w ere also contaminated and began to turn yellow at the time of harvest. They were consequently omitted from the table of data. There were a f ew nodules found in their roots. There were ohly two nodules found in the jars inoculated with Radiobacter and these were used as checks. The complete plants were harvested and ost of the sand was removed by wasning the roots. It was impossible, however, to remove all the srnd without losing part of the roots, and therefore some of the sand remained on the roots. The plants from each jar were placed in separate cheese-cloth bags and were carefully labeled. The bags containing the plants were placed in an electric oven and the plants dried at C. The plants were weighed and their dry weight was recorded . ■ ■ . ' . ' * _S- The seed coats and cotyledons that were collected during the period of growth of the plants were added to their respective jar of dry plants and were in- cluded in the total dry weight. Method of Analysis . The total plants from each jar were digested with 35 c. c. concentrated nitrogen-f ree 5 ijrams of ITa^SgO^ 0.2 grams of CaSOd in 800 c. c. Kjeldahl flasks until the solution was clear and no trace of undigested organic matter was present. The sand that remained on the roots of the plants was removed during the decantation of the digested solution and was placed in separate dishes. The sand was dried and weighed ana the weights were subtracted fror their respective original dry weights of the cowpeas. This gave the exact total dry weight of the plants. The digested solution was ua&e up to 500 c. c. in 500 c. c. measuring flasks. One tenth, or ^0 c. c., of this solution was taken for analysis. This portion was poured into Kjeldahl flasks and the solution was made up to 2p0 c. c. with ammonia- free water. Excess concentrated alkali (KOH) solution was addeu arid a few granules of zinc. These solutions were distilled over as IJH-j in 1 mg. HC1 solution. The excess of acid J was neutralized ty 0.5 mg. alkali (.KOH) arid the total amount of nitrogen was thus determined. The results of the analysis are shown in Table 1. Experiment 1 was partly repeated (April 11 - May 31 , 1322) in one part glass jars with the following inoculation: Lira bean, cowpea, beggarweed, Azotobacter and Had lob uo ter organisms. The plants in the check jars and those in the jars inoculated with Azotobacter arid Kacliobactar organisms began to turn yellow on the sixth week and a few days later, most of the leaves dropped. The other jars remained green and grew .uch taller than the others. There was no difference in the ether jars as far as growth was concerned. Time aid not per- mit for the analysis of these plants, but they confirm the first part of the experiment as far as behavior of the plants during growth is concerned. -s- Eesults of Experiment T The results of this experiment show that the plants inoculated with peanut bacteria gave the highest amount of nitrogen fixed. Those inoculated with ccwpea, beggarweed, and partridge pea (Cassia) run ail of about the sa .e nitrogen content, and those inoculated with Rndiobacter served as checks. In the latter case, it shows that where the organism did net lead tc nodule forma- tion, the plant 8 did not derive any benefit. The larger fixation found with the peanut bacteria ay have been due to these plants being less injured by the mildew. It is significant, however, that they should be as good or better than plants inoculated with cowpea bacteria. EXPERIMENT II. Cross-Inoculation cf Garden Peas (Alaska) as Influencing Fixation. Tr.is experiment was conducted tc study the effect of cross- inoculation of garden pea with vetch organisms, also the efficiency of different pea culture The procedure .and experimental methods were the sa .e as that in the exp eriment I. The seeds were planted October ZB, 1321. Twelve seeds were planted in each jar. The plant food was applied in the same manner as in experiment 1. As far as could bs observed, there was no difference in growth. The height of the plants varied somewhat, but that was also true of plants in the same jars. The plants were harvested December 22, 1021. The checks were contaminated, but since there were no checks necessary they did net alter the results of the experiment. 1/Thile harvesting, great care was taken in observing the root development, the number of pods formed, and the average height of the plants. There was hardly any difference in root development in all cf the jars. There was some difference in the number cf pods developed, however, the number ■ . . ' . -Q- j of pods are recorded for each jar in Table 2. Tne process cf analysis was the sa.-e as that employed in the experiment with the cowpeas, vith the exception that the plants were dried in squall tin boxes. There were 20 jars inoculated. The cultures used were transfers from Dr. A. L. Whiting's stock cultures, two were obtained from Dr. E. B. Free of the Wisconsin Station. There were l4 Jars representing 7 cultures of garden pea bacteria of different cultural history, and 4 jars of vetch representing two cultures of widely different cultural history. The inoculation was rather heavy. One bottle was used for each jar and the cultures were cf the sane age. The treatments, as well as the analysis cf the plants and other data, is given in Table 2. Results of Experiment II While there is a difference in the average total nitrogen present in the plants that were inoculated with the two pea and vetch cultures in favor of the vetch organise, it cannot be said that this would have been true had there been the same number of jars inoculated in both cases. However, of the jars inoculated with garden pea bacteria, four might be taken, such as 23, jO, and 40, the average of which would almost be tne sa. e as the four vetch jars. In a problem of this nature, there are other factors that enter in the results. One of these is the age of the cultures. Cultures of different age will have different effects upon the plants. The condition of tne growth of the plant at the time the organism is at its highest efficiency would be another factor. In some cases, a plant may not be in condition to use all of the nitro- gen produced by the organism. One or all of the factors may be ^.resent in some cases and therefore give results which would lean one to believe that one organism ha 3 a better effect than another organism in cross- inoculation. It would require mere data in order to draw conclusions of a reliable ' . . - - 10 - nature, but the oross-inoculat icn in this ex_.eri.ient proved as good or better than the inoculation cf peas with pea bacteria. - 12 - PART IT Nitrogen Assimilation "by Non-legumes Through tae Action of Nodules and Free Living Nitrogen Fixing Bacteria. Experiment 3 was planned to determine whether oats could benefit hy the presence of nodule and free living nitrogen- f ixing bacteria. Tne import nee of the problem is at once apparent for the greatest problem in American agriculture would be solved if the cereal crops c.uld obtain nitrogen sufficient for their growth from nitrogen-f ixing bacteria living in the soil. REVIEW OF THE LITERATURE The literature dealing with nitrogen assimilation by non- legumes through the presence of nitrogen-fixing bacteria is more extensive than that dealing with the chemical efficiency of cross- inoculation vs. inoculation with bacteria common to a given legume. T vc decidedly different views have been ^ resented regarding the conditions that should exist for the non-legume to benefit by the presence of the nitrogen- fixing bacteria. These views are very simply stated as follows: (l) That nodules should be produced on the roots of plants in order for a benefit to 'be derived, ( > ) that nodule production is entirely unessential in order to p*er it a non- legume to ass iaai late the nitrogen fixed by the bacteria. The investigations of Schneider, Burrill and Hansen, Stutzer, Burri end Maul, Grosbusch and Lee; eraaann, were all concerned with the thought that nodule production was essential for success. They were supporters of the first view. It should be emphasized here, however, that so-**e of these workers, while failing to produce nodules, did however record observations indicating that in their judgment the growth of the non- legumes was beneficially influenced by the presence of some of the bacteria. Schneider, working under the late Dr. Burrill at this station in 1233, . - 13 - cult i vat ed nodule bacteria froi masse lus Vnlg.mris upon bean extract agar, then upon a fixture of bean extract and corn extract agar, and finally upon pure corn root extract agar. Transfers were made every sixth day. Aft-r the cultures had grown for a no nth up on the pure corn root extract, they "ere ap-. lied upon germinating seed of corn aaxd oats. Though the inoculated corn plants produced no nodules, Schneider claimed that they were more thrifty than the unincculated plants. He described and figured the infection of some of the root hair cells as well as some of the epidermal and parenchi.nl cells. No effect was noticed upon oats. Burrill and Hansen took up the problem where Schneider left it. They attempted to inoculate tomato seedlings vith PseudO'-enas Iladi ci cola and sweet clover organises. They found no abnormal conditions in their roots, but the plants that were inoculated were mere thrifty in every case. Other cases in this direction have been reported. Stutzer, Burri and haul inoculated mustard plants with nodule bacteria which had gradually become accustomed to a custard plant medivna, but without success. Grosbusch experimented with C-ram inae but his results were negative. Lemuermann studied the difference in nutrition between the Leguminosae and the Ora-:- in.ee. He believed that the reason for the existence of bacterial symbiosis in the Leguminosae and not in the Ora, mans are, namely, the smaller transpiration current, the higher acidity cf the root sap, and the greater root develop/ ent of the former as compared with the latter. The second view is supported by the striking results reported by H . V. Joshi, Agricultural Research Institute, Pusa, India. His conception of the benefit derived by non- legumes is based upon the fact that nitrogen fixation as such is a function cf certain bacteria and it will proceed regardless of the nature of the plant present. Any plant requiring nitrogen should be able to assimilate the product of these bacteria. . TA’SL'S III - Pronin r results obtained 'y •;. y. JohM with corn, oats, and wheat. - 15 " He conceived the idee, while he was conducting a cross- inoculation experi- ment with peas, math, and grab* inoculated with two strains of cowpea organises and arhar, dhaincha, and gokarn c rgani sms . When he exa; i ned the roots of the plants tnat were inoculated, although there were no nodules present, still their roots appeared much ~ore vigorous than those of the uninoculated, the inoculation of the organ is..; appearing to exercise a stimulating effect. He then suggested t nat nitrogen night he fixed in the sand independent to the plant. Upon this he based his hypothetical assumption that the nitrogen thus fixed must he equally available to any kind of ^lant, whether belonging to legnminc sae cr not. Also if one kind of nitrogen-fixing organism, could bring about increased growth, another organism* like Azotubacter ay be able to show the e;ae effect. The re- sults of his experiments with corn, oats, and wheat, besides the legumes, show marked differences in total nitrogen. The difference of growth is also noticed in the photographs which accompany his work. EXPERIMENT III. Oats Grown in the Presence of Nodule and Free-Living N i t ro g en- Fixing Bacteria. The procedure of this experiment was the same as in the previous experi- ments, The amount and rate of giant food was the sane as in experiments I and £ with the addition of two applications of 1C c. c. of . annite to jars 44 to ?4 inclusive, and CaClIO-Tp 43^0 v/as ad led to jars 55 and ^ 6 . The aannite solution ✓ was made up by adding 5 grams of ..annite in 200 c. c. of nitrogen-free distilled water. The oats was planted ilarch 23> 1922. There were 15 seeds planted in each jar. There were no differences noticed on the plants until April 4. At this time the jars treated with Ca(NO*)o were best and jars 47 and 48 were a trifle better than the rest. The other jars vere about the sane. From this time on, jars 42 to 54 inclusive turned yellow and remained so. The stems of -lo- the plants in these jars were very thin in comparison with the plants in jars 55 and About May 3» some of the plants on the check jars and also in the jars treated with bacteria started to seed. On ’■'ay S, all of the plants treated with organisms and the control jars seeded. Jars 55 and 56 received 70 c. c. of Ca(l!0-)2 each or equivalent to 41.51 H. A picture of the plants was taken May 11, 1922. The total plants were harvested May 16, 1922. The method of analysis was the same as that employed in experiments 1 and 2 with the exception that the digested solution was made up to 200 c. c. and one-half of the solution was taken for analysis. The results of the analysis are shown in Table 3 . Results of Experiment III The results of the total nitrogen determinations indicate in eight out of ten cases, no gain whatever to the oats for the nitrogen that may have been fixed by the bacteria. In the case of jar numbers 4? and 52, a larger increase is evident, but unfortunately the number of plants was not definitely known and therefore it cannot be said it was a gain due to the presence of the bacteria or to increased number of plants. Judging from the percent of nitrogen, it might easily be ascribed to on increased number of plants. If oats could benefit from the presence of these organisms, there should have been more nitrogen contained in she eight jars than found in the checks. . . . I p II I I I . -17- table V . Dry weights and total nitrogen content of oats growing in the presence of Badiobacter, Azotobacter, and the bacteria cf sweet clover, garden peas., aad co wg eas . Jar numb or Treatment Cult -tors number ’eight cf 1 lants Dry weight in gins. Total N ter . J&r _ _ 43 Check C*£S • 7 O 2.15C 15.4 44 tt 3^ £.080 15.6 43 Badiobacter 1 %~ r 30 1.550 14.6 46 V 140 £3 2.300 13.2 4? Azotobacter 135 35 £.290 21.0 4S tt 1T& 30 2 . 02 C 15.4 43 Sweet clover 144 34 2.230 15.6 50 tt tt 141 32 1 .S 30 « H 51 Garden peas 145 30 1. 820 13.0 52 r n 146 33 3 . l 60 23.3 53 Cowpeas 1.49 30 2.150 14.2 54 n 150 62 > 2.540 13.6 55 Ca(NO 4-KpO J - *- 44 4.600 47.0 56 tt 50 5.110 55.0 -IS- Oats growing in the presence of nodule and free living nitrogen-fixing bacteria. - 19 - SmiAEY The results of experiments 1 and £ shc.v that there is no disadvantage in cross- inoculation and it night he of an advantage, as shown in the case of the performance of the peanut inoculation in experiment 1 and the vetch in exp: eri»i-ent2. The results of experiment 3 show that the oats do not derive any benefit by the presence of' the nodule and free- living nitrogen-f ixing b. cteria. The results do net support the results obtained by N. V. Joshi. It would require ere work than reported herein to settle either one of these questions. ACENOTOEDGIJMT The author takes this opportunity to express his gratitude to Professor A. L. Yhiting under whose direction and careful guidance this work was conducted. -20 bibliography 192 ? • Burr ill and Hansen. Is syub i sis possible between legume bacteria and. no n- legu -e p lant s? Ills. Exp. St a. Bui. 202. 1907- Grosbusch. Rhizobiuir. radio ico la. T Jsu. Inang. Biss. (Bonn) 1920. Josfci. Studies on the root nodule organ! s of the leguminous plants. Memoir of the Dept, of Agr. of India, Vol. 1, No. 9- ISIS. Koch and Butler. Cross- inoculation of legu es. Soil Science 6:397-403, N • IS. 100 4. Lee. .eraann. Unersuchungen ueber eigine Ernahrungsunterichiede der Leguninosen und Grandneen und ihre wahrscheiniiche Ursache. Verhandl. Gesell . Deut . Nettvrf. u. Aerzte (19C4), , II, 1, l45 • Landw. Vers. Stat. 1904, 67 , 207-51. 1921. Lohiiis arxl Hansen. Nodule bacteria of leguminous giants. Jour. Agr. Res. Vcl. XX, No. 7, 1921. 1893 . Schneider. A new factor in economic agriculture. Ills. Agr. Exp. Sta. Bui. 29, (1893), 3 OI- 3 I 9 . 1913. Spratt. A comparative account of the root nodules of the Leguainosae. Annals of Botany, p. 18$. IS 96 . Stutzer, Burri, and Maul. Untersuchengen ueber anpassungswer.ogen von Bacillus radicicola an einen frenden Hahrboden. Centbl. Bakt. 2 Abt. 2, 065 - 669 . 1920. Whiting and Schoonover. ' Nitrogen fixation by cowpeas and nodule bacteria Soil Science Vcl. X, No. 6 , Bee. 1920. 1920. Whiting .end Hansen. Cross- inoculation studies with the nodule bacteria of li:ta beans, navy beans, cowpeas and other of the cowpea group. Soil Science, Vol. X, No. 4, Oct. 1920. I