,,university ,,of ,,illinois ,agricultural ,experiment ,station4 ,,bulletin ,,no4 #id4 ,,nitrogen ,,bacteria ,,and ,,legumes 7,,with ,,special ,,reference ,,to ,,red ,,clover1 ,,cowpeas1 ,,soy ,,beans1 ,,alfalfa1 ,,and ,,sweet ,,clover1 ,,on ,,illinois ,,soils74 ,,by ,,cyril ,g4 ,,hopkins4 ,work and ,knowledge are a stronger team than ,work and ,work4 ,illustration ,,urbana1 ,,illinois1 ,,february1 #aijd4 ,,summary ,,of ,,bulletin ,,no4 #id4 #a4 ,soil nitrogen cannot be used by plants until it is changed to the form of nitrate nitrogen by the nitrifying bacteria4 ,page #cjg #b4 ,atmospheric nitrogen cannot be used by any agricultural plants1 excepting legumes1 and even leguminous plants have no power to obtain nitrogen from the air unless they are provided with the proper nitrogen-gathering bacteria4 ,page #cji #c4 ,as a rule each important agricultural legume must have its own particular species of bacteria4 ,page #caa #d4 ,the frequent failure of red clover in normal seasons1 especially on normal soils occupying the highest land1 is undoubtedly due in part at least to the absence of the proper bacteria 7sometimes the soil lacks lime or phosphorus74 ,page #cac #e4 ,on the very acid soils1 where clover has never been grown successfully1 applications of ground limestone should be made where legumes are to be grown4 ,page #cae #f4 ,cowpeas need not be inoculated1 because the cowpea bacteria are usually either present in the soil or are introduced with the seed in sufficient numbers to effect a good degree of infection if the soil is suitable and if cowpeas are seeded upon the same land for two successive years4 ,page #cag #g4 ,cowpeas grown on infected soil on the ,university of ,illinois soil experiment field contained four times as much nitrogen as the same kind of cowpeas grown on similar land which was not infected4 ,page #cai #h4 ,as a rule soy bean fields should be inoculated when first seeded to soy beans1 otherwise they may be grown on the same land for three or four years before the soil becomes thoroughly infected4 ,page #cbj #i4 ,investigations1 reported in this bulletin1 furnish conclusive proof that infected sweet clover soil can be used for inoculating alfalfa fields and with the same results as are obtained when the infected soil is obtained from an old alfalfa field4 7,sweet clover is a tall1 rank-growing1 sweet-scented leguminous plant widely distributed over ,illinois1 especially along the roadsides in the northern and central parts of the state1 and in many places in bottom lands in southern ,illinois47 ,page #cbd #aj4 ,this bulletin will be sent free of charge to any one interested in ,illinois agriculture upon request to ,e4 ,davenport1 ,director ,agricultural ,experiment ,station1 ,urbana1 ,illinois2 and1 if so requested1 the name of the applicant will be placed upon the permanent mailing list of the ,experiment ,station1 so that all bulletins will be sent to him as they are issued4 ,conclusions ,page #cbg ,,nitrogen ,,bacteria ,,and ,,legumes 7,,with ,,special ,,reference ,,to ,,red ,,clover1 ,,cowpeas1 ,,soy ,,beans1 ,,alfalfa1 ,,and ,,sweet ,,clover1 ,,on ,,illinois ,,soils74 ,,by ,,cyril ,g4 ,,hopkins1 ,,chief ,,in ,,agronomy ,,and ,,chemistry4 ,among the several different classes or groups of bacteria there are two which are of special importance to agriculture because of their relation to the element nitrogen1 this being commonly considered the most valuable element of plant food4#a ,these two classes of bacteria are1 first1 the nitrifying bacteria1 and1 second1 the nitrogen-gathering bacteria4 ,,the ,,nitrifying ,,bacteria4 ,the nitrifying bacteria are those which have the power to form nitrates4 ,in the following brief discussion of this subject we include at least three species of bacteria which by their combined or successive action have the power to transform organic nitrogen into nitrate nitrogen1 which is a suitable form of nitrogen for plant food4 ,for the exact information which we now have regarding the nitrifying bacteria we are indebted to the researches of ,pasteur and ,schlsing and ,mntz of ,france1 ,winogradsky of ,russia1 ,warington of ,england1 and others4 ,the nitrogen in the soil is almost entirely in organic compounds2 that is1 the nitrogen 7which is a gas in the free1 or uncombined1 state7 is united or combined with other elements1 notably with carbon1 hydrogen1 and oxygen1 in the form of partially decayed vegetable or organic matter4 7,by organic matter we mean matter which has been formed by the growth of some organism1 either plant or animal1 as grass or flesh47 ,plants cannot use the free nitrogen of the air as plant food1 neither can they use the organic compounds of nitrogen which occur in the soil4 ,there are at least three different kinds of bacteria1 and also three different steps or stages involved in the process of nitrification1 the nitrogen being changed from the organic compounds first into the ammonia#b form1 second1 into the nitrite form1 and third into the nitrate form4 ,during the process the nitrogen is separated from the carbon and other elements composing the insoluble organic matter1 and is united or combined with oxygen and some alkaline element 7as calcium7 to form the soluble nitrate1 such as calcium nitrate1 which is one of the most suitable compounds of nitrogen for plant food4 ,calcium is the alkaline element contained in lime or limestone4 ,the name calcium nitrate indicates just what elements this compound contains2 namely1 calcium1 nitrogen1 and oxygen4 7,in the names of compounds the ending -ate always means oxygen47 ,this is the general process of nitrification in which the nitrifying bacteria transform or transfer the nitrogen from insoluble organic compounds into soluble compounds in which it may serve as available plant food4 ,the nitrate which is thus formed may be calcium nitrate or magnesium nitrate or potassium nitrate or even sodium nitrate1 depending upon which of these alkaline elements is present in the must suitable form4 ,if no alkaline element is present in available form then no nitrates can be made in the soil4 ,one of the reasons for applying ground limestone to soils which are deficient in lime is to furnish the element calcium in suitable form for the formation of nitrates in the process of nitrification4 ,ground limestone is calcium carbonate 7,ca,c,o71 a compound containing one atom of calcium 7,ca71 one atom of carbon 7,c7 and three atoms of oxygen 7,o74 ,this is the same form of lime which is contained naturally in limestone soils--soils which are noted for their great productiveness--and it is generally the most economical form of lime to use for correcting soil acidity and promoting nitrification4 ,in the process of nitrification1 that is in the formation of nitrates1 there is required1 not only the presence of calcium1 or some other alkaline element1 in suitable form1 but also a good supply of the element oxygen2 for calcium nitrate1 ,ca7,,no71 contains one atom of calcium 7,ca71 two atoms of nitrogen 7,n71 and six atoms of oxygen 7,o71 in each molecule as indicated in the formula1 ,ca7,,no74 ,magnesium nitrate1 ,mg7,,no71 potassium nitrate1 ,,kno 7,k is from the ,latin word ,kalium1 which means potassium71 and all other nitrates1 also1 contain oxygen4 ,the supply of oxygen for the formation of nitrates in the soil comes from the air1 which consists of about twenty percent oxygen1 seventy-eight percent nitrogen1 and two percent of other elements and compounds1 as argon1 carbon dioxid1 ,,co1 water vapor1 ,h,o1 etc4 ,one of the important effects of cultivation1 or tillage1 is that it permits the air more freely to enter the soil1 and thus promotes nitrification4 ,,the ,,nitrogen-,,gathering ,,bacteria4 ,as stated above1 the nitrogen naturally in the soil is contained almost entirely in the organic matter4 ,any process which tends to decompose or destroy this organic matter1 such as nitrification or other forms of oxidation1 will also tend to reduce the total stock of nitrogen in the soil4 ,because of this fact the matter of restoring nitrogen to the soil becomes of very great importance4 ,of course a part of the nitrogen removed in crops may be returned in the manure produced on the farm2 and nitrogen may also be bought in the markets in such forms as sodium nitrate 7containing #ae to #af percent of nitrogen71 ammonium sulfate 7containing #bj to #ba percent of nitrogen71 and dried blood 7containing #ab to #ae percent nitrogen72 but1 when we bear in mind that such commercial nitrogen costs about #ae cents a pound1 and that one bushel of corn contains about one pound of nitrogen1 it will be seen at once that the purchase of nitrogen cannot be considered practicable in general farming1 although in market gardening1 and in some other kinds of intensive agriculture1 commercial nitrogen can often he used with very marked profit4 ,nitrogen is removed from the soil not only in the crops grown1 but also1 and frequently in larger amounts per annum1 in the drainage waters1 and in some other ways1 as by denitrification and by the blowing and washing of the surface soil4 ,professor ,snyder1 of the ,minnesota ,experiment ,station1 has shown that during a series of years the total loss of nitrogen from some ,minnesota soils in some cases amounts to several times the amount actually used in the crops produced4 ,considering all of these facts1 and the additional facts that there are about seventy-five million pounds of atmospheric nitrogen resting upon every acre of land1 and that it is possible to obtain unlimited quantities of nitrogen from the air for use of farm crops1 and at very small cost1 the inevitable conclusion is that the inexhaustible supply of nitrogen in the air is the store from which we must draw to maintain a sufficient amount of this element in the soil for the most profitable crop yields4 ,it is often stated that leguminous plants1 such as clover1 have power to obtain free nitrogen from the air4 ,this is not strictly true4 ,red clover1 for example1 has no power in itself to get nitrogen from the air4 ,it is true1 however1 that the microscopic organisms#c which commonly live in tubercles upon the roots of the clover plant do have the power to take free nitrogen from the air and cause it to unite with other elements to form compounds suitable for plant food4 ,the clover plant then draws upon this combined nitrogen in the root tubercles1 and makes use of it in its own growth1 both in the tops and in the roots of the plant4 ,these nitrogen-gathering bacteria live in tubercles upon the roots of various leguminous plants1#d such as red clover1 white clover1 alfalfa1 sweet clover1 cowpeas1 soy beans1 vetch1 field-peas1 garden-peas1 field and garden beans1 etc4 ,these tubercles vary in size from a pinhead to a pea1 varying with the different kinds of plants1 being especially small upon some of the clovers1 and very large upon cowpeas and soy beans4 ,the tubercles are1 of course1 easily seen with the eye1 but the tubercle is only the home of the bacteria1 somewhat as the ball upon the willow twig is the home of the insects within4 ,the bacteria themselves are far too small to be seen with the unaided eye1 although they can be seen by means of the most powerful microscope4 ,several million bacteria may inhabit a single tubercle4 ,it is not necessary to see the bacteria1 because if we find the tubercles upon the roots of the plant1 we know that the bacteria are present within1 as otherwise the tubercle would not be formed4 ,although the plant itself1 as clover1 for example1 has no power to feed upon the free or uncombined nitrogen in the air1 yet these nitrogen-gathering bacteria do have the power to absorb the free nitrogen and cause it to combine with other elements1 forming nitrates or other compounds which are suitable forms of nitrogen for plant food4 ,it has also been demonstrated that1 as a rule1 there are different species of nitrogen-gathering bacteria for markedly different species of leguminous plants4 ,thus we have one kind of bacteria for red clover1 another kind for cowpeas1 another kind for soy beans1 and still a different kind for alfalfa4#e ,,the ,,red ,,clover ,,bacteria4 ,that clover has no power in itself to gather atmospheric nitrogen1 and that the bacteria do have power to feed the clover plant with nitrogen gathered from the air is very easy to demonstrate4 ,it is one of the regular laboratory practices of the students in soil fertility in the ,agricultural ,college to make this demonstration4 ,plate #a is an illustration of such student work4 ,the two pots which are shown were provided with all elements of plant food1 excepting the one element nitrogen4 ,thus far the two pots are exactly alike4 ,each contains no nitrogen1 as indicated by the label 8,no ,n40 ,each pot is planted with the same number of red clover seeds4 ,to the right-hand pot1 however1 some bacteria 78,bac407 were added1 while none were added to the left-hand pot4 ,these bacteria were obtained by taking about one pound of soil from a clover-field where abundance of tubercles were found on the clover roots1 adding this soil to about one quart of pure water1 shaking for a few minutes1 allowing the soil to settle1 then taking a small quantity of almost clear solution1 and adding it to the pot which we wished to inoculate with the red clover bacteria4 ,aside from the addition of these microscopic bacteria to the right-hand pot1 these two pots were treated exactly alike throughout the experiment4 ,it will be plainly seen that where the bacteria were added the clover was furnished with sufficient nitrogen to make a strong and luxuriant growth1 while without the bacteria the clover 7in the left-hand pot7 only germinated and made what little growth it could with the small amount of nitrogen contained in the seed4 ,this result is the difference between success and failure of the clover crop4 ,illustration3 ,,plate #a4 ,,red ,,clover3 ,,effect ,,of ,,bacteria4 ,,no ,,nitrogen ,,in ,,the ,,soil ,,of ,,either ,,pot4 ,in general the clover bacteria are well distributed over the northern and central part of ,illinois1 but we now have some very strong evidence that they are not well distributed in some soils of large area in southern ,illinois4 ,there is also some evidence that they were not originally present even in the soils where they are now found in great abundance2 and1 furthermore1 it seems very probable that these bacteria may cease to live in a soil where they have once been present1 provided clover is not grown on the land for several years4 ,it will help us to understand this matter if we bear in mind that the home of these bacteria is the tubercle upon the clover root4 ,it is quite evident that they will continue to live upon the decaying tubercles or roots for three or four years after the clover plant has been killed4 ,on the other hand1 we have some notable evidence that the bacteria do not continue to live in a soil after five or six years' continuous cropping with absolutely no clover growing on the land during those years4 ,it is a simple matter for any one to determine whether the bacteria are present or not1 for the tubercles which are formed if the bacteria are present are plainly seen attached to small roots4 ,they look somewhat like miniature potatoes1 varying in size from pinheads on clover to peas on soy beans or cowpeas4 7,see ,plates #b and #d47 ,it is important to remember that the bacteria live in the soil and not in the seed4 ,when clover is cut for seed1 it is frequently left to lie upon the ground until the straw becomes half rotten and very dirty2 and1 consequently when it is threshed1 it practically always happens that there is at least some small amount of dust and dirt taken with the seed4 ,this dirt is almost sure to carry with it some bacteria from the soil4 ,if these few bacteria are scattered with the clover seed when it is sowed they will inoculate at least a few plants1 and if they are allowed to multiply on these plants1 and especially if the same field is repeatedly seeded with clover1 the soil will ultimately become thoroughly infected with the clover bacteria4 ,of course they may be carried from one part of the farm to another1 or even from one farm to another1 by various agencies1 as dust or wind storms1 surface drainage or flood waters1 manure made from clover hay1 implements used in cultivating the soil1 etc41 etc4 ,many of the older farmers of ,illinois have stated to the writer that when this country was very new it was commonly found difficult to get a 8catch0 of clover on new land4 ,after a good 8catch0 was once gotten1 then it was easier to get clover to grow on that land the next time4 ,there was a saying among the farmers that clover would not do well until they got the 8wild nature0 out of the land4 ,their final success was undoubtedly due1 not to getting anything out of the land1 but rather to getting the bacteria into the land4 ,several ,illinois farmers have reported some quite remarkable results from very light applications of the clover chaff or straw 7obtained in hulling clover7 in its beneficial effect on clover on land where it was otherwise difficult to get a 8catch40 ,there is a somewhat general belief among farmers of long experience that clover straw or chaff has some special value in getting a catch of clover aside from its value as manure or for the seed which it sometimes contains4 ,manager ,f4 ,a4 ,warner of the ,sibley ,estate1 ,ford ,county1 recently stated to the writer that they had had very great difficulty to get clover to grow when they first began growing clover on that large estate1 some six or eight years ago1 although1 after a good crop was once secured1 they rarely had any further difficulty in getting a catch of clover on the same land4 ,on the common gray prairie soil of the ,lower ,illinoisan ,glaciation1 in southern ,illinois1 the commonest type of soil in more than twenty counties1 practically no red clover is grown4 ,in the spring of #aijc we seeded red clover on that type of soil in three places2 namely1 on the ,university of ,illinois soil experiment fields near ,edgewood1 ,effingham ,county1 near ,du ,bois1 ,washington ,county1 and near ,cutler1 ,perry ,county4 ,on certain plots the soil acidity had been corrected with lime and an abundant supply of phosphorus 7in bone meal7 had been provided1 potassium also having been added on some plots4 ,these fields were carefully examined the latter part of ,june1 and at ,cutler and ,du ,bois the clover was found to be dead or dying1 and no tubercles could be found upon the clover roots1 although on the clover which had been seeded at about the same time on the ,university fields at ,urbana the root tubercles were found in great abundance4 ,at ,edgewood a few tubercles were found and the clover appeared to be growing fairly well4 ,infected red clover soil was at once procured and scattered over the fields at ,edgewood1 ,du ,bois1 and ,cutler1 but it was evidently too late to be of any marked benefit4 ,at ,cutler and at ,du ,bois the clover was a complete failure4 7,it will be tried again next year47 ,at ,edgewood it continued to grow fairly well1 and its progress next season 7#aijd7 will be watched with much interest4 ,it should be stated that the ,experiment ,station has been growing clover for several years with varying degrees of failure on land adjoining the present clover field at ,edgewood1 and it is possible that this year's apparent success from the start is due in part at least to the bacteria which have been incidentally introduced and multiplied year after year and scattered over the adjoining land by wind and dust storms4 ,before the close of the season the tubercles developed in abundance on the roots of the clover at ,edgewood4 ,an experience reported by ,professor ,herbert ,w4 ,mumford1 of the ,animal ,husbandry ,department of this university1 will be of interest and value in this connection4 ,professor ,mumford commonly grows clover in his rotations on his own private farm1 but he states that at one time one particular field was cropped continuously with timothy1 oats1 and corn for some six years or more without any clover whatever4 ,it was then again seeded to clover1 but the crop made a complete failure1 although on other land where clover had been grown more recently a successful clover crop was grown from the same kind of seed seeded at about the same time4 ,the following year this particular field was again seeded to clover4 ,this time the 8catch0 was not a total failure1 but it was too poor to save1 and it was plowed up and the land again seeded to clover the next year1 and an excellent catch of clover resulted4 ,after this1 clover was frequently grown on this field1 and no special difficulty was had in getting good crops4 ,while the failure of clover may often be due to drouth1 and in some places due to soil acidity 7lack of lime71 and sometimes even due to an insufficient supply of available phosphorus or of potassium1 we now know with certainty that it sometimes fails because of the absence of the nitrogen-gathering bacteria1 especially on land which has never grown clover1 and probably also on land which has not grown it recently4 ,we should always remember that the bacteria do not thrive in strongly acid soils4 ,even though they may sometimes live in such soils and perhaps produce some tubercles upon the roots of certain hardy1 strong growing legumes1 like cowpeas1 nevertheless we are obtaining some strong evidence that in such acid soils they have but little power to gather nitrogen from the air4 ,that ground limestone is the most economical and satisfactory material to use in correcting the acidity of soils is strongly indicated by the information we have thus far obtained4 ,on the upland prairie soils of the ,lower ,illinoisan ,glaciation where red clover has never been grown successfully1 largely because of the acidity of the soil1 it will undoubtedly be helpful and profitable not only to correct the acidity of the soil with ground limestone1 but also to secure infected soil from some field of timber land or bottom land where red clover is growing1 well provided with root tubercles1 and inoculate the field with it4 ,this soil should be collected to a depth of three or four inches and scattered over the prairie land at the rate of a few hundred pounds per acre at the time the clover is seeded or before4 ,,the ,,cowpea ,,bacteria4 ,plate #b is made from a photograph of a cowpea root with the tubercles upon it4 ,this illustration shows the cowpea tubercles at nearly natural size1 which is about as large as the seed of ordinary garden peas4 ,illustration3 ,,plate #b4 ,,cowpea ,,root ,,tubercles1 ,,natural ,,size4 ,the cowpea bacteria are already quite widely distributed in southern ,illinois1 especially where this crop has been grown for several years1 but they are not common in the soils of other parts of the state4 ,it is doubtful1 however1 if it is necessary or even worth while to take the trouble to inoculate soil for cowpeas4 ,some few tubercles almost invariably develop on cowpea roots the first year they are seeded1 even where they have never been grown before1 and if seeded the second year on the same land the plants are usually abundantly provided with root tubercles4 ,just why the cowpea bacteria develop so rapidly even without special inoculation is not definitely known4 ,it may be that the same bacteria also live on some other leguminous plant which is more or less widely distributed over the state1 but it seems more likely that the bacteria are brought with the seed4 ,as a matter of fact1 the cowpea harvest is usually dirty4 ,this is an annual plant1 and consequently the crop is grown on recently plowed land and is sometimes cultivated during the season4 ,cowpeas are commonly harvested with a mowing machine and then raked up on the loose ground4 ,when they are threshed more or less dirt remains with the seed4 ,furthermore1 the seed coats are not infrequently cracked1 thus providing an excellent place for the lodgment of particles of soil4 ,whether it would be profitable to inoculate the land for cowpeas would depend very largely upon the difficulty or cost of obtaining the infected material4 ,if soil thoroughly infected with the cowpea bacteria can be scattered over the land at the rate of about #b1jjj pounds to the acre at a cost of #a4jj or less per ton1 it might prove profitable4 ,it is doubtful if a light application of #ajj or #bjj pounds would produce any very marked effect in the yield the first season4 ,after the soil becomes well infected the cowpeas then obtain much nitrogen from the air1 and the yield of cowpeas is likely to be largely increased4 ,of course there is no fixation of atmospheric nitrogen if there are no tubercles on the roots4 ,in #aijb several plots of cowpeas were seeded on the soil experiment field at the university4 ,one of those plots 7#djd7 had become thoroughly infected with the cowpea bacteria because of its being so situated that more or less surface drainage water flowed over it from an adjacent field upon which cowpeas had been grown for three successive years4 ,another plot 7#djh71 owing to a slightly different situation1 had not become infected4 ,the two plots were seeded in ,july after a crop of oats had been removed from the land4 ,within three weeks after seeding1 numerous root tubercles could be found on the plants on the infected plot4 ,later on1 ten average consecutive plants were taken up as completely as possible1 and #dab tubercles were found on the roots1 making an average of more than #dj tubercles to the plant4 ,on ,plot #djh only an occasional plant was found infected1 and such plants would usually have only a single large tubercle on their roots4 ,ten average plants not infected were collected from ,plot #djh for comparison with the ten infected plants from ,plot #djd4 ,illustration3 ,,plate #c4 ,,cowpeas2 ,,effect ,,of ,,bacteria ,,in ,,ordinary ,,illinois ,,black ,,prairie ,,soil4 ,plate #c shows these two bunches of plants1 the infected plants with root tubercles on the right1 and the plants without tubercles on the left4 ,four more sets of ten plants each were then collected1 two sets from ,plot #djd and two from ,plot #djh4 ,each set of infected plants was separated into three parts1 7#a7 tops1 7#b7 roots1 7#c7 tubercles2 and each set of plants not infected was separated into 7#a7 tops1 and 7#b7 roots4 ,all of these samples were dried and analyzed for nitrogen4 ,the results obtained are shown in ,table #a4 ,the results clearly show the very great value of the nitrogen-gathering bacteria in growing cowpeas4 ,in each of the separate trials ,a1 ,b1 and ,c1 the infected plants contained about twice as much total dry matter as the plants not infected4 ,the infected plants also contained a much higher percent of nitrogen than the plants not infected1 the infected plants containing #d4ji to #d4cc percent in the tops and #a4de to #a4ec percent in the roots1 while those not infected contained only #b4cb to #b4fi percent in the tops and 4#hh percent in the roots4 ,besides this1 the tubercles on the infected plants contain #e4gf to #f4je percent of nitrogen4 ,in these young and rapidly growing plants the tubercles are much richer in nitrogen than any other part of the plant4 ,it should be stated that as the plants approach maturity the nitrogen is largely absorbed from the tubercles and stored in the tops and roots4 ,at the time these plants were taken up the tubercles actually contained more nitrogen than the roots4 ,the infected plants contained nearly four times as much nitrogen as the plants not infected1 and about three-fourths of the total nitrogen in the infected plants was obtained from the air4 ,the roots and tubercles of the infected plants contained six to seven times as much nitrogen as the roots of the plants not infected4 ,,table ,i4--,,fixation ,,of ,,nitrogen ,,by ,,cowpeas4 ,,cowpea ,,plants4 ,dry ,nitrogen,nitrogen,nitrogen ---------------------------- matter1content1amount1 fixed by ,no4 ,part cgs4 percent4 cgs4 bacteria1 cgs4 ---------------------------------------- --------------------- ,a#a--,ten plants1 ,tops #cehj #d4ji #adf with bacteria ,roots #fbj #a4de #i present4 ,tubercles #aij #e4ig #aa ---- --- ,total #dcij #aff #abe ------------------------ ,a#b--,ten plants1 ,tops #aefj #b4db #ch without bacteria ,roots #cjj 4#hh #c ---- --- ,total #ahfj #da ---------------------------------------- --------------------- ,b#a--,ten plants1 ,tops #cigj #d4ca #aga with bacteria ,roots #fij #a4dg #aj present4 ,tubercles #cjj #f4je #ah ---- --- ,total #difj #aii #adj ------------------------ ,b#b--,ten plants1 ,tops #bjfj #b4fi #ee without bacteria ,roots #dcj 4#hh #d ---- --- ,total #bdij #ei ---------------------------------------- --------------------- ,c#a--,ten plants1 ,tops #ccjj #d4cc #adc with bacteria ,roots #ebj #a4ec #h present4 ,tubercles #bij #e4gf #ag ---- --- ,total #daaj #afh #abd ------------------------ ,c#b--,ten plants1 ,tops #agcj #b4cb #dj without bacteria ,roots #djj 4#hh #d ---- --- ,total #bacj #dd ---------------------------------------- --------------------- ,,the ,,soy ,,bean ,,bacteria4 ,soy bean bacteria are evidently much less likely to be carried with the seed than are the cowpea bacteria4 ,the soy bean plant grows more erectly than the cowpea 7see ,circular ,no4 #fi1 8,the ,cowpea and ,soy ,bean in ,illinois071 and the crop is quite commonly harvested with a self-binder which keeps it quite free from dirt4 ,the soy bean seed is nearly round and smooth1 and the seed coat is not commonly cracked4 ,these facts may explain why the soy bean seed carry so few bacteria as compared with cowpeas4 ,on one of the soil experiment fields on the university farm at ,urbana1 where soy beams have been grown for three years1 no tubercles could be found on the plants either the first or second year1 and only an occasional plant with tubercles could be found the third year4 ,in #aijb a series of plots1 some of which had been treated in different ways with applications of limestone1 phosphorus1 and potassium1 were seeded with soy beans4 ,no tubercles could be found at any time during the season on the soy beans growing on any of the different plots4 ,in #aijc the same plots were again seeded to soy beans1 and at the same time part of each plot was inoculated with infected soy bean soil drilled in with the seed at the rate of about #ejj pounds of infected soil to the acre4 ,when the plants were only a few weeks old tubercles were to be found upon many plants growing where the infected soil had been applied1 and before the close of the season at least half of these plants in the inoculated part of the field had one or more tubercles upon their roots1 and some plants could be found whose roots were abundantly provided with tubercles4 7,see ,plate #d47 ,on the uninoculated part of the field soy bean plants were examined probably fifty times during the season1 several plants being taken up each time1 but not a single tubercle was found at any time1 notwithstanding that this was the second crop of soy beans upon this soil4 ,of course the inoculated part of the field did not become sufficiently infected to markedly benefit the #aijc crop1 but it is planned to grow soy beans upon this field again in #aijd when the bacteria will doubtless have multiplied sufficiently to produce marked results in the growth of the crop4 ,from these and from other somewhat similar experiments it is concluded that as a rule soy beans should be inoculated when they are first seeded1 and that they should then be grown a second year upon the same land4 ,if soy beans are afterward grown upon this land once in every three or four years1 the soil will doubtless remain well infected with the soy bean bacteria4 ,it is believed that #ajj pounds of infected soy bean soil per acre will be sufficient to produce a thorough infection the second year1 and it is improbable that one ton of infected soil per acre would produce a thorough inoculation the first season4 ,one ton is only twenty times #ajj pounds1 while one tubercle which will be produced during a single season from a single bacterium may contain many million bacteria1 thus it will be seen that it will be more economical to inoculate rather lightly and allow the bacteria to multiply themselves rather than to inoculate heavily at great expense4 ,illustration3 ,,plate #d4 ,,soy ,,bean ,,root ,,tubercles1 ,,natural ,,size4 ,it may be stated that the infected soy bean soil used in these experiments was obtained from ,mr ,a4 ,a4 ,hinkley of ,du ,bois1 ,illinois1 who has been growing soy beans on the same land for many years until it has become well infected4 ,mr4 ,hinkley has consented to furnish infected soy bean soil so far as he is able to do without serious interference with his regular work1 to any one who may desire it1 at a price which will cover his expense and loss4 ,this will probably amount to about #a4jj for the first #ajj pounds and fifty cents for each additional #ajj pounds1 in the shipment1 including the cost of bags1 the purchaser to pay freight from ,bois station1 which is located in ,washington ,county1 ,illinois1 on the ,illinois ,central ,railroad4 ,,the ,,alfalfa ,,and ,,sweet ,,clover ,,bacteria4 ,that soil inoculation with alfalfa bacteria is commonly of very great value in growing alfalfa has been shown very conclusively by the investigations reported in ,bulletin ,no4 #gf1f 8,alfalfa on ,illinois ,soil40 ,in some places1 however1 inoculation was found to be unnecessary4 ,a careful and extensive investigation of alfalfa growing in different parts of ,illinois revealed the fact1 as stated in ,bulletin #gf1 8that the alfalfa bacteria are certainly present in some places in the state while in most other places they are certainly not present in sufficient number to become of appreciable assistance to the alfalfa within three or four years1 and the question naturally arises how it happens that some fields are already infected while others are not40 ,it was suggested in that bulletin that the alfalfa bacteria may 8live on some other plants besides alfalfa and that one of these plants is native or has been introduced in certain sections0 of the state4 ,it was also suggested 8that a few bacteria are always carried with alfalfa seed1 and that if the alfalfa is grown continuously or repeatedly in any place the soil will finally become thoroughly infected1 and the bacteria will then be carried by flood waters1 dust storms1 etc41 over adjoining fields1 and possibly for long distances1 especially along river valleys40 ,this latter suggestion was known to be a fact at the time it was written2 and subsequent investigations have furnished conclusive proof that the alfalfa bacteria do live upon another plant2 namely1 the ordinary sweet clover 7melilotus alba74 ,this is a rank-growing leguminous plant1 frequently reaching a height of four to six feet4 ,when young it markedly resembles alfalfa1 but it can easily be distinguished by its characteristic odor when cut or bruised1 as by rubbing between the hands4 ,as the sweet clover approaches maturity it differs very much from alfalfa4 ,the sweet clover grows very tall1 and usually branches from a main stem4 ,it has white flowers 7there is also a less common yellow variety71 and the seeds are borne in small round pods 7usually containing only one or two seeds each71 arranged on long slender spikes1 each spike bearing many pods4 ,the alfalfa commonly grows about two and a half feet high1 with many stems growing from the crown of the root1 especially after it is two or three years old4 ,it bears purple flowers and peculiar spiral-shaped seed pods4 ,sweet clover is a biennial plant1 dying after reaching maturity1 which commonly occurs the second year of its growth4 ,like many other biennial plants1 it probably often lives more than two years if not allowed to produce seed4 ,alfalfa is a perennial plant1 and it is said that there are alfalfa fields which have been cut annually for more than fifty years without reseeding4 ,the similarity of alfalfa and sweet clover when young1 and also the similarity of the tubercles formed on the roots of each have long been noticed1 and the possibility of the same bacteria living upon both plants has already been suggested in the agricultural press4 ,during the season of #aijc the writer spent some time in the northern part of ,illinois in connection with the general and detail surveys of ,illinois soil4 ,many new fields of alfalfa were observed1 and they were carefully examined for root tubercles4 ,in ,winnebago ,county1 where sweet clover is very prevalent along roadsides and in waste places1 it was noted that the abundance of root tubercles on the alfalfa plants seemed to be closely related to the presence of sweet clover in the vicinity1 strongly indicating that the bacteria which live upon sweet clover were also at home upon the alfalfa roots4 ,these indications were strengthened by further investigations in ,lake ,county1 especially upon the ,fowler farm1 near ,lake ,villa1 where a field of alfalfa seeded last spring without artificial inoculation was found to be thoroughly infected with the bacteria1 and growing vigorously with a good dark green color4 ,this field had a few sweet clover plants growing in it1 and the borders of the field were covered with sweet clover4 ,other fields of alfalfa seeded in the neighborhood at the same time1 but upon soils where sweet clover had not grown near by1 were apparently complete failures1 many of the plants having died and most of those still living being only a few inches high1 very weak1 and yellow or pale green in color4#g ,in order to obtain more absolute knowledge regarding this important subject1 a series of pot culture experiments has been carried on under controlled conditions in the pot culture laboratory at the university4 ,five pots were filled with sterilized sand which was practically devoid of plant food4 ,a supply of phosphorus1 potassium1 and all other mineral elements necessary for the growth of plants was added to each of the five pots1 care being taken to keep the sand practically free of combined nitrogen4 ,alfalfa seed were then planted in each of the five pots1 and at the same time four of the five pots were inoculated as follows3 ,pot ,no4 #a4--,not inoculated 7check pot74 ,pot ,no4 #b4--;,inoculated with bacteria obtained from infected alfalfa soil4 ,pot ,no4 #c4--;,inoculated with bacteria obtained from alfalfa root tubercles4 ,pot ,no4 #d4--;,inoculated with bacteria obtained from infected sweet clover soil4 ,pot ,no4 #e4--;,inoculated with bacteria obtained from sweet clover root tubercles4 ,plate #e clearly shows the results obtained and certainly furnishes conclusive proof that the same effect is produced upon the growth of the alfalfa whether the nitrogen-gathering bacteria used for the inoculation are obtained from alfalfa soil1 from alfalfa tubercles1 from sweet clover soil1 or from sweet clover tubercles4 ,it also illustrates the importance of bacteria in growing alfalfa as will be seen by comparing the four inoculated pots with the uninoculated pot1 which is ,no4 #a1 on the left in each series of views4 ,the upper view was taken when the alfalfa plants were five weeks old2 the next series when they were six weeks old2 the next1 seven weeks old2 and the lower series when they were eight weeks old1 from the time of seeding4 ,a duplicate series of pots prepared in exactly the same manner gave similar results4 ,the infected alfalfa soil was obtained from a field of three-year-old alfalfa1 which was inoculated when first seeded1 with infected alfalfa soil obtained from an old alfalfa field in ,kansas4 ,about one pound of this soil was shaken in a quart of water1 the soil allowed to settle1 and some of the nearly clear solution used for the inoculation of ,pot ,no4 #b4 ,the alfalfa tubercles from which bacteria were obtained were carefully washed in distilled water to free them from adhering soil particles1 and then rubbed up in distilled water1 a small amount of this water being then used for the inoculation of ,pot ,no4 #c4 ,the infected sweet clover soil was obtained from a place by the roadside where sweet clover was growing luxuriantly and well provided with root tubercles4 ,this place was about two miles from the nearest field ever seeded to alfalfa1 so far as known4 ,a water extract from this soil was used to inoculate ,pot ,no4 #d4 ,the bacteria from sweet clover tubercles were obtained in the same manner as those from alfalfa tubercles1 and were used to inoculate ,pot ,no4 #e4 ,illustration3 ,,plate #e4 ,,alfalfa3 ,,effect ,,of ,,bacteria ,,from ,,alfalfa ,,and ,,from ,,sweet ,,clover4 ,pot #a4--,no bacteria4 ,pots #b and #c4--;,bacteria from alfalfa4 ,pots #d and #e4--;,bacteria from sweet clover4 ,the four series of photographs were taken five1 six1 seven and eight weeks from time of planting1 respectively4 ,from these investigations we thus have conclusive evidence that infected sweet clover soil can be used for the inoculation of alfalfa fields1 the bacteria of the two plants acting the same4 ,the infected soil may be obtained from any place where the sweet clover is found growing with abundance of tubercles on its roots4 ,the soil may be collected to a depth of three or four inches and scattered over the alfalfa field at the rate of #ajj pounds or more to the acre4 ,it is well to scatter the infected soil at about the time the alfalfa is seeded1 and harrow it in with the alfalfa seed1 although it may be applied some days or even some weeks before seeding time1 and probably it would be all right to apply the infected soil the fall before1 for it is known that the bacteria will live in soil for several months1 even though the soil be placed in sacks and allowed to become quite dry4 ,investigations have shown that #ajj pounds of thoroughly infected soil to the acre is sufficient to produce a very satisfactory inoculation within one year from the time it is applied4 ,of course1 somewhat heavier applications may well be made if it can be done at small expense4 ,the infected soil need not be applied with any high degree of uniformity1 but special care should be taken that the higher places and watersheds are not missed in scattering it over the field4 ,if a few square yards1 or even square rods1 should be missed on the slopes or lower land1 it would make but little difference1 as the bacteria will be washed over such places from the higher land4 ,after the soil becomes somewhat dry it is easily scattered by hand from the wagon or from a sack which one can carry4 ,sometimes it is applied by means of an end gate seeder or a fertilizer drill1 or it could be spread by a manure spreader with an application of manure4 ,the question naturally arises whether there is not danger of getting some sweet clover seed with the infected sweet clover soil1 and thus of getting sweet clover mixed with the alfalfa in the field4 ,in the writer's opinion there is little or nothing to fear in this matter4 ,in the first place1 the amount of sweet clover seed thus obtained would be very small1 probably none at all1 if one were careful to scrape off the vegetable matter1 and perhaps a half inch of earth before collecting the infected soil 7most of the bacteria are probably between one-half inch and six inches in depth1 as most of the tubercles develop and decay between those depths72 second2 it is doubtful if a small amount of sweet clover hay would lessen the value of alfalfa hay in the least1 for stock frequently eat small amounts of sweet clover of their own choice even when it is nearly mature1 and if it is cut while still quite immature and tender it makes quite satisfactory hay1 so much so that in some sections of the ,united ,states1 particularly in the ,south1 sweet clover is regularly seeded on fields and cut for hay1 and it is found to be a valuable and very nutritious feed1 the live stock eating it in large quantities1 and with apparent relish1 after they have acquired a taste for it2 third1 sweet clover is not known as a bad weed in the fields or meadows1 even where it has been a common roadside plant for many years1 and1 being naturally a biennial plant1 if it were cut down every five or six weeks1 as we commonly cut alfalfa during the season1 it would almost certainly die out after a few years while alfalfa1 a perennial plant1 would continue to live4 ,only one instance has come to the writer's attention where alfalfa has been growing for several years with sweet clover growing in the field or fence rows beside it4 ,this is on the farm of ,mr4 ,d4 ,s4 ,mayhew1 of ,mercer ,county1 ,illinois1 who writes as follows regarding the matter3 8,will say that the sweet clover has made no headway in my meadow1 as it did not go to seed1 on account of my cutting it so often4 ,the sweet clover got into the alfalfa in the seed when ,i sowed it4 ,i do not think it will do any harm in a meadow1 but ,i believe it would do harm in a pasture if it wasn't cut down as stock will not eat the sweet clover40 ,of course if sweet clover should get into the field and persist in growing1 and if it were found to injure the alfalfa appreciably or markedly1 we can always resort to plowing the ground up and growing corn or other crops1 thus obtaining some benefit from the leguminous crop for its fertilizing value1 and at the same time completely eradicating the sweet clover1 but leaving the soil well infected with alfalfa bacteria ready to serve in case alfalfa should be again seeded within a few years4 ,,conclusions4 ,in general agriculture in ,illinois1 whether it be grain farming or ordinary livestock farming1 the growing of legumes is absolutely essential as a part of any economic system which shall maintain the fertility of the soil2 and for the successful growing of legumes the presence and assistance of the proper species of nitrogen-gathering bacteria are also absolutely essential4 ,these facts being granted1 it certainly follows that when sowing any legume on land1 where the same legume has never been grown before1 or perhaps where it has not been successfully grown within recent years1 we should always consider the matter of inoculation2 and1 unless there is good reason to believe that the soil has been inoculated by the washing from other higher lying land where these bacteria are known to be present or by applications of manure made from that legume1 or by some other such incidental means2 or unless there is evidence that the bacteria are carried with the seed in sufficient quantity to effect a satisfactory inoculation 7as appears to be the case with the cowpea71 then we should inoculate the soil directly with the specific bacteria required by the legume which we desire to grow4 ,while some ,illinois soils are becoming deficient in phosphorus and in lime1 especially in the southern part of the state1 and while phosphorus#h and ground limestone can be applied to such soils with marked benefit and profit1 especially for the growing of legumes1 there is abundant evidence that one of the dominant causes for the failure or unsatisfactory growth of some of our most valuable legumes1 and on some soils the sole cause of failure1 is the absence of the proper nitrogen-gathering bacteria4 ,there is no reason to believe that any of the different species of nitrogen-gathering bacteria will live in the soil for more than a few years#i in the entire absence of any legume upon which they naturally live1 and the accumulating evidence strongly indicates that the bacteria which are present in places in our soils1 such as the red clover bacteria1 now found abundantly in many places in the state1 especially in northern and central ,illinois1 the cowpea bacteria more common in southern ,illinois1 and the alfalfa or sweet clover bacteria1 which are becoming prevalent in some sections--that all these have been1 and are being1 gradually introduced and extended almost entirely by mere chance4 ,of course if the wagon-wheel1 which carries the mud along the road1 carries with it sweet clover seed from one place to another1 it may also carry the sweet clover bacteria which live on the sweet clover roots4 ,it now seems absurd to suppose that there were red clover bacteria in ,illinois soil before red clover itself was grown on ,illinois soil1 unless the same bacteria live also upon some other legume which was native to our soils4 ,there is some evidence that the vetch bacteria are native to our soil1 possibly living upon the native wild vetches4 ,at any rate1 tubercles commonly develop on vetch roots without artificial inoculation4 ,investigations are in progress to ascertain whether the notorious failure of crimson clover in ,illinois may not be due in part1 at least1 to the absence of the proper bacteria4 7,it has been stated by some writers that the bacteria of crimson clover and those of red clover are identical1 but we already have some reason to doubt the accuracy of this statement47 ,,footnotes #a ,it should be remembered that there are ten essential elements of plant food each of which is of equal importance to the plant1 for if the plant is deprived of any one of the ten essential elements it is impossible for it to develop and mature4 ,carbon has no market value as plant food because the plant obtains carbon in the form of carbon dioxid1 a gas which is present everywhere in the atmosphere and which the plant inhales through its leaves4 ,both hydrogen and oxygen are without market value because they are the elements which compose water1 a liquid compound which plants absorb through their roots4 ,calcium1 magnesium1 iron1 and sulfur have no market value as elements of plant food because they are present in practically all soils in abundance as compared with the amounts required in plant growth4 ,the three elements nitrogen1 phosphorus1 and potassium1 do have market values1 because they are required by plants in very considerable quantities1 and they are present in most soils in rather limited amounts1 and when the available supply of any one of these elements becomes too much reduced in a soil the crop yield also becomes reduced4 ,for further information regarding the use of these elements of plant food on ,illinois soils1 see ,circular ,no4 #fh1 8,methods of ,maintaining the ,productive ,capacity of ,illinois ,soils40 #b ,technically this first step is preliminary to1 and not a part of1 nitrification4 #c ,among the scientists who were prominent in making these discoveries regarding the action of bacteria in the fixation of atmospheric nitrogen were ,hellriegel1 ,willfarth1 and ,nobbe in ,germany1 ,atwater in ,america1 ,lawes and ,gilbert in ,england1 and ,boussingault and ,ville in ,france4 #d ,it may be well to call attention to the fact that there are numerous instances where two different kinds of plants live together in intimate partnership relation4 ,if only one of the two plants receives benefit from this relationship or association1 then the plant receiving the benefit is celled a parasite4 ,thus the mistletoe is a parasite upon the elm or gum or other tree on which it lives4 ,the mistletoe draws its nourishment from the tree4 ,the tree is injured rather than benefited by the mistletoe4 ,dodder is also a parasitic plant1 living upon other plants1 except during the early part of its growth4 ,ticks and lice are common examples of animal parasites living upon other animals4 ,in some cases a relationship exists which is not parasitic but symbiotic4 ,the term symbiosis1 which is commonly used by biologists to define this relationship1 means living together in mutual helpfulness4 ,the association of bees and flowers may serve to illustrate this mutual helpfulness1 although this is not an example of intimate symbiosis4 ,thus the bees obtain their food from the flowers and1 in turn1 the flowers1 many of them1 are incapable of producing seed or fruit unless the pollen is carried from the male flower to the female flower by bees or other agencies4 ,it is well known that plant lice and ants are mutually helpful4 ,likewise the association of nitrogen-gathering bacteria and leguminous plants is a relationship of mutual helpfulness and this is one of the best illustrations of what is meant by symbiosis4 ,the legume furnishes a home for the bacteria and also furnishes in its juice or sap most of the nourishment upon which the bacteria live4 ,the bacteria1 on the other hand1 take nitrogen from the air contained in the pores of the soil1 and cause this nitrogen to combine with other elements in suitable form for plant food which is then given up to the legume for its own nourishment4 ,another illustration of remarkable parasitism1 if not1 indeed1 one of true symbiosis1 is found in the common lichens living upon rocks and trees4 ,the lichen is not a single plant1 but two plants--an alga1 which lives upon the wood or stone1 and a fungus which lives upon the alga4 ,alg also live in the free state separate from fungi1 and the present opinion of botanists seems to be that when the two are associated in the form of lichens this association is not detrimental1 but rather beneficial1 to the alga1 as well as to the parasitic fungus4 ,if this is true1 then it is another case of true symbiosis4 7,there is reason to believe that the fungus has some power to feed upon atmospheric nitrogen1 and then to furnish combined nitrogen to the alga upon which it lives47 ,in the symbiosis of leguminous plants and nitrogen-gathering bacteria we have a partnership or relationship of immeasurable value to agriculture4 ,here is a class of plants 7legumes7 that are capable of consuming or utilizing nitrogen in quantities larger than could possibly be obtained from ordinary soils for any considerable length of time4 ,they have no power in themselves of taking nitrogen from the atmosphere1 and to them the symbiotic relation with this low order of plants 7the nitrogen-gathering bacteria7 is especially helpful1 and for the best results it is absolutely necessary4 #e ,there are some noteworthy exceptions to this rule 7see following pages for illustration71 and there is some evidence that1 by a comparatively long process of breeding1 or evolution1 the bacteria which naturally live upon one kind of legume may gradually develop the power to live upon a distinctly different legume to which they were not at first adapted4 ,of course this process of forcing bacteria to live upon a legume to which they are not naturally adapted has little or no practical value because it is unnecessary if there is a species of bacteria which naturally lives upon the same legume4 ,on the other hand1 if1 by any such process of breeding1 or evolution1 a species of nitrogen-gathering bacteria could be developed which could live on a non-leguminous plant1 as corn1 for example1 it would be of incalculable practical value4 ,as yet the efforts of bacteriologists1 working on this problem1 have given only negative results1 so far as known to the writer4 #f ,in this connection attention is called to the fact that the so-called 8spot disease0 of alfalfa1 which is not uncommon in the western states1 especially during wet seasons1 became somewhat prevalent in ,illinois in #aijc4 ,when the effect of this disease becomes marked1 the leaves turn yellow and growth is retarded4 ,if this occurs the alfalfa should be clipped4 ,this is the only effective remedy known to be practicable4 ,seeding alfalfa with a light nurse crop is gaining favor in ,illinois4 #g ,some of these observations have already been reported in the agricultural press4 7,see1 for example1 the ,breeders' ,gazette1 ,september #i1 #aijc1 page #cia1 and ,september #af1 #aijc1 page #ddb47 #h ,steamed bone meal is the most economical and satisfactory form of phosphorus for use on ,illinois soils1 unless ground rock phosphate 7not acid phosphate7 shall prove to be still more economical4 ,experiments to determine this are in progress4 #i ,just how long the bacteria will live in a soil without a leguminous crop upon which they can feed is not definitely known4 ,certainly they live for two or three years1 but probably not more than five or six years4 ,further investigation is needed to establish the length of time the different kinds of bacteria may remain in the soil under different conditions4