H, { . S. DEPARTMENT Ol BUREAU OF PLANT INDUSTRY- BUL: B. T. (. 11.1.1'HAV, Chiei AGRICULTURE. NO. 72, PARI IV. ,/■ /SG-i. INOCULATION OF S NITROGEN-FIXING BA BY A. F. WOODS, Acting Chief op the Bureai oi Plant Industry. WASHINGTON: GOVERNMENT PRINTING OFFICE. 1905. B. I". I— KJ3. INOCULATION OF SOIL WITH NITROGEN- FIXING BACTERIA. INTRODUCTION. The publication of the. results obtained with pure cultures in inocu- lating leguminous plants has resulted in a very greal demand being made upon the Department of Agriculture for inoculating material. The distribution made during L904 was for the purpose of obtaining ;i large number of tests of the method under average farm condition-, and it was impossible to anticipate the demand which has arisen this spring (1905), the total quantity prepared for spring distribution having been promised early in February. It is expected, however, that thi- fall and next springs further distribution will be made as far as our limited facilities will permit. Statements to the effeel that the Depart inent has -topped the distribution of these culture-, are therefore erroneous. Applications for future distributions should state what legume is to be ■-own. time of --oh ing, and quantity of seed to be treated. THE COMMERCIAL, PRODUCTION OF CULTURES. The patent which the Department of Agriculture holds upon the met hod of grow ing and disl ributing these organisms was taken out in such a way that no i can maintain a monopoly of the manufacture of 9uch culture-, li is held in the name of Dr. George /. M c, who developed and perfected the met li.nl. a- described in former publications. Upon application the Department furnishes without discrimination all necessary information, and a- far a- possible " start- ing" or Eoundat ion culture--, to the bacteriologists representing experi- ment stations and commercial concerns uhieh claim to 1m- properly equipped, Gut it doe- not in any wa\ guarantee their product. It is not likelj that persons without expert knowledge can successfully multiply cultures <>i' these organisms for -ale or distribution, and it is understood thai an\ cultures furnished are to he treated according to the methods devised l>_\ tin Department. 3 4 NITROGEN-FIXING BACTERIA. Before experimenting with any bacterial preparations for legumes, the farmer should study thoroughly the soil conditions under which the use of cultures offers any possibility of gain." Briefly, these conditions may be summed up as follows: WHEN INOCULATION IS NECESSARY. Inoculation is necessary — (1) On a soil low in organic matter that has not previously borne leguminous crops. (2) If the legumes previously grown on the same land were devoid of nodules, or " nitrogen knots," showing the need for supplying the nodule-forming bacteria. (3) When the legume to be sown belongs to a species not closely related to one previously grown on the same soil. For instance, soil in which red clover forms nodules will often fail to produce nodules on alfalfa when sown with alfalfa for the first time. WHEN INOCULATION MAY PROVE ADVANTAGEOUS. Inoculation may prove advantageous — (1) When the soil produces a sickly growth of legumes, even though their roots show some nodules. If the cultures introduced are of the highest virility, their use will often result in a more vigorous growth. (2) When a leguminous crop already sown has made a stand, but gives evidence of failing, due to the absence of root nodules. The use of the culture liquid as a spray or by mixture with soil and top-dressing may save the stand if other conditions are favorable. WHEN INOCULATION IS UNNECESSARY. On the other hand, inoculation is unnecessary and offers little jwos- ■pect of gain — (1) Where the leguminous crops usually grown arc producing up to the average and the roots show nodules in normal abundance. Cultures of nitrogen-fixing bacteria are not to In regarded in the light of fertilizers, increasing yields under all average conditions. They do not contain the nitrogen itself, but the bacteria make it possible for the legumes to secure nitrogen from the air (through the forma- tion of root nodules), and where the soil is already adequately supplied with these bacteria it will not usually pay to practice any form of artificial inoculation. (2) When the soil is already rich in nitrogen. It is neither necessary nor profitable to inoculate a soil rich in nitro- gen when sowing legumes. Not only does the available nitrogen in « Fully described in Farmers' Bulletin No. 214 of the Department of Agriculture, which will be sent without cost upon application to the Secretary of Agriculture. COST OF < I 1.111:1 I) the soil render the formation of nodules less necessary, bul nit rogenous materials in the soil largely prei » ■ 1 1 1 the bacteria from forming nodules. Am increased virility in nitrogen-fixing power possessed by any t\ pes of bacteria yel distributed may be rapidlj losl in a soil contain- ing an abundance of nitrogen, because the bacteria are rapidly multi- plying in a medium in which there is no premium on vigor in securing atmospheric nitrogen. WHEN FAILURE IS TO BE EXPECTED. Inoculation will fail where other conditions (aside from the need of bacteria) are not taken into account, as the following: (1) In -oil thai is acid and in need. of lime. Liming to correct acidity is as important for the proper activity of the bacteria as for the growth of the plants. (2) En soil that responds in a marked way to fertilizers, such as potash, phosphoric acid, or lime. The activity of the bacteria in securing nitrogen from the air ami rendering it available to the legumes doe- not do away with the need for Such fertilizing elements as potash and phosphorus. (3) It must also be remembered that inoculation does not "act like magic;" it will not overcome results due to had seed, improper preparation and cultivation of ground, and decidedly adverse condi- tion- of w eat her or climate. In the use of cultures, also, failure is a hue- 1 certain where the direc- tions are not carefully studied and intelligently followed. (4) As the physics, the chemistry, and the biolog} of soils are studied in the laboratory and by means of actual field-plot trial- to determine yield and quality of crops and the effect of one crop on the following crop-, the ven great complexity of soil and farm manage- ment becomes more manifest. The value of pure-bred bacteria, whether associated with the crop or existing independently in the soil, as is true of fertilizers, can not lie predicted u ith certainty on any soil \\ ithout trial. Success on simi- lar near-by lands may he taken as good evidence. But, unlike fer- tilizer-, bacteria should in time We so inexpensive that each farmer can afford to try them for each leguminous crop on each field or soil t\ pe on hi- farm. The method- of distributing in dried form and the easy methods of multiplying on the farm in sufficient quantities to inoculate fields will make it possible to have all fields inoculated at all time-. COST OF CULTURES. The question of the proper price for the commercial product is caus- ing considerable inquirj among prospective experimenters and i- of importance. The expenses which a commercial concern must neces- sarily meet, such a- rent, heat, light, insurance, postage, advertising, 6 NITROGEN-FIXING BACTERIA. etc. , aside from laboratory assistance and clerical hire, make airy com- parison with the cost to the Government of similar cultures difficult. The statement that the cultures cost but a few cents an acre refers only to the raw materials which make up the package. It is more than probable that natural competition will considerably reduce the present valuation of the commercial product, and the wisdom of patenting the Department's methods to prevent the formation of a monopoly is already demonstrated. INCREASING CULTURES. We are receiving numerous requests from persons who have secured commercial cultures, as well as those sent out from the Department of Agriculture, for information as to the methods employed in producing a large quantity of liquid culture from the dry culture secured as a starter; that is, how to make an "acre culture' 1 do for 25 or 100 acres. Such methods will give good results only when special pre- cautions are taken, and on this account have not been generally recom- mended. The contaminations, such as yeasts, molds, etc., which are bound to occur to a greater or less extent, are apt to take possession of the culture solution in which the bacteria are being multiplied, and unless great care is taken in thoroughly sterilizing all utensils em- ployed the resulting culture will have no beneficial effect. The extra time required to secure sufficient growth of bacteria in 10 gallons of solution from a dry culture originally intended to produce a 1-gallou liquid culture makes the risk from contamination much greater than where the dry culture is proportioned in size to the larger amount of solution. If a growth sufficient to cloud the solution takes place within two days, the chances of securing an efficient culture are much better than where a longer time is taken; so that the volume of solution prepared should never exceed the actual requirements of the occasion. The following directions are based on making 10 gallons of liquid culture, sufficient to inoculate 20 bushels of seed. By a little compu- tation the directions may be adapted to 5 gallons or to any intermedi- ate quantities. PREPARING AND USING THE CULTURE SOLUTION. To prepare the culture solution, first select the tub, bucket, or other vessel in which you wish to grow the bacteria. Clean and scald it out thoroughly. For making the culture solution, rain water that has been thoroughly boiled and allowed to cool is best, though any good drinking water will answer. Add to 10 gallons of water 12 ounces of either brown or granulated (preferably granulated) sugar, 14 ounces of potassium phosphate (monobasic), which can be obtained at any drug store, and one-sixteenth ounce (30 grains) of magnesium sulphate. * KEEPING CI LTURES FOR FUT1 R] USE. ( Stir until dissolved, then carefully open the small package containing the bacteria-laden cotton and drop the cotton into the solution. I><> doI handle any more than is absolutely necessary. Cover the tub with a moi -i , clean cloth toproteci from dust, mold spores, etc. Keep in a warm place, but never let the temperature rise above Mood heat. A.fter twenty four hours add 6 ounces of ammonium phosphate and allow the mixture to -land for another twenty four hours. The Liquid should now be cloudy and ready for use; if sufficient growth has not taken place to bring about this cloudiness, further tune should be given, not to exceed .1 few daj s. To inoculate seed. Use enough culture liquid to moisten the seed thoroughly aboul one half of a gallon per bushel. This inoculating may he done either in a tub ^v trough, or by sprinkling the culture liquid on the seed on a clean floor and stirring and turning the heaps of seed with shovels until all are thoroughly moistened. After inoc ulation the seed should he spread out in a clean, shady plaee until sufficiently dry to handle. If planting is not to he done at once, the seed must he thoroughly dried to prevent molding. In dry weather aboul 25 bushels can he dried in half a day on .'inn square feet of floor spaee. To do this there must lie several open windows or doors to allow a free circulation of air, and the seed must be frequently stirred with a lawn rake. The inoculated seed, if thoroughly dried, may usually be kept without deterioration for several months. '/',, inoculah soil. Take enough dry earth or sand so that the solu- tion will merely inoi-ten it. The -nil should he preferably from the field to he inoculated, so a- to avoid spreading diseases or weed-. Mix thoroughly, so that all the particles of soil are moistened. Thoroughly mix this earth with four or five times as much; spread this inoculated -oil thinly and evenly over the prepared ground exactly a- if spreading fertilizer. The inoculated soil should he har- rowed in immediately to protect the bacteria from sunlight. In using this method allow l gallon of the Liquid culture to 1 acre- or less. Either <'\' the methods described may he used, a- may he most convenient. '/'■■ prevent any possible delay, tfu necessary chemicals should he ordered in advanc< . If the local druggist does not have them in stock, he .-an doubtless secure them within a reasonable time. KEEPING CULTURES FOR FUTURE USE. The question is frequently arising as to the possibility of the farm- er'- keeping over cultures from one year to another by soaking up a little of the lit pi id culture in cotton and drying this coi 1 1 m. Tins pro- posed practici is not tobt advised hi any case. Contaminations take place so readily, an tarted spread so rapidly, that for assured 8 NITROGEN-FIXING BACTERIA. good results it is absolutely necessary to start with a pure culture. The pure culture, moreover, can only be prepared by a trained bac- teriologist with laboratory facilities. These cultures in the dry state will keep, under ordinary conditions, from six months to a year. There is an additional reason, fully as important, which makes the above method impracticable. The cultivation of the bacteria for any considerable length of time in solutions containing ammonium salts rapidly lessens their infective power and their ability to gather nitro- gen from the air, so that transfers or new cultures made with absorb- ent cotton from the cultures prepared for field use would contain organisms of reduced efficiency. It is partly owing to these factors that it is impracticable to distribute the bacteria in liquid cultures and maintain the requisite effectiveness. In the use of cultures for inoculating soil the farmer should be guided, as in all other matters pertaining to soil treatment, by his own peculiar needs and should not give too great weight to the experiences of others whose soil conditions may differ widely. It would be unwisi to invest largely in any new method for increasing plant growth, whether bacterial or of any other nature, i •p/'thout previously experi- menting in a small way. DANGER OF INOCULATION BY SOIL TRANSFER. Satisfactory inoculations have been obtained by transferring soil from old fields on which the legume has been grown, but experience has shown that there are dangers incident to such methods of soil transfer which it is wise to avoid. The source of supply of such soil should be very definitely known, and in no case should soil be used from fields which have previously borne any crops affected with a fungous disease, a bacterial disease, or with nematodes. Where a rotation of crops is practiced, it is often difficult to make sure of this factor, so that the method of soil transfer is, under average circumstances, open to suspicion, if not to positive objection. Numerous animal and plant parasites live in the soil for years, and are already established in so many localities that it is mani- festly unwise to ship soil indiscriminately from one portion of the country to another. The bacterial diseases of the tomato, potato, and eggplant, and the club-root, brown-rot, and wilt disease of the cabbage, all more or less widely distributed, are readily transmitted in the soil; while in the South and West there are the wilt diseases of cotton, melons, sweet potatoes, cow peas, and flax, and various nematoid and root-rot diseases which might easily become a serious menace over areas much larger than they now occupy if deliberately spread by the careless use of soil for inoculation purposes. There are several insect and fungous diseases of clover to be avoided, and various diseases of beans and peas. There is also a disease of alfalfa, the ••leaf spot."" which is IT Kl ■< I I.I I Rl [NOOl |. \TlnN. 9 causing damage in some regions. These arc only a few of many dis eases liable to be transmitted in soils. The farmer should therefore be on his guard. The danger from such sources is l>\ no means imaginary. The Department of Agriculture has had specific cases of such accidental distribution reported, and if the business of selling soil tor inoculation is made to Sourish by farmers purchasing without question "alfalfa -oil." "cowpea soil," etc., there is every reason to believe thai experience will demonstrate the folly of such haphazard methods. Of scarcely less importance is the danger of disseminating noxious weeds and insect pests through thi^ plan of inoculation by mean- of Boils. Even though weeds may not have been serious in the first field, tlic great numbers of dormant seeds requiring but a slight change in surroundings to produce germination are always a menace. The enor- mous damage to crops caused by introduced insects and weeds should convey a warning and lead to caution. It is not the part of good judgmenl to view the risk as a slight one justified l>\ the end in view. PURE-CULTURE INOCULATION. The extensive experiments carried on by the Department of Agri- culture during L904 demonstrated the fact that, by the proper use of pure cultures, the nodule bacteria are actually carried into the soil in such a way a- to form root nodules, and where other condition- are favorable the inoculation thus brought about makes possible the growth of each legume in soils where it had previously failed from the lack of bacteria. The original cultures used, however, must be prepared with the utmost care and with a view to preserving and increasing their natural power as " nitrogen fixers" rather than merely to make them grow under favorable conditions. The methods devised in our Laboratory of Plant Physiology are based on well-recognized principles of plant breeding and -election, and mark a decided advance in the production of culture- for soil inoculation. The old pure- culture method- were not effective, for reasons clearly stated by Dr. Moore in Bulletin No. 71 of the Bureau of riant Industry and by Dr. Moore and Mr. Robinson in Farmers' Bulletin No. 21 t. The Department of Agriculture is continuing the work of develop- ing types of the bacteria associated with leguminous plants, which will have greater activity, collecting from the air more nitrogen per acre than forms now common in nature or available from laboratories. It i- desirable that similar investigations should be conducted with reference to the nitrogen-fixing bacteria existing in the soil independ- ent of the legumes. Important steps have already been taken along this line, but the very large demand for cultures for leguminous crops, by consuming the time ^>\' the laboratory force, has seriously retarded these investigations during the past year. 10 NITROGEN-FIXING BACTEKIA. The Department is reacty to cooperate with experiment .stations and commercial firms, to give and to receive suggestions, to test the prod- uct of others, and to furnish, as far as possible, cultures to be tested in the laboratory and under field conditions. There is nothing in the nature of the processes involved which would prevent a competent bacteriologist, after some experience in this particular field, from producing cultures of as high a grade as those sent out by the Department, and every assistance will be given to competent persons desiring to undertake the work. A. F. Woods, Acting Chief, Bureau of Plant Industry. Approved: James Wilson, Secretary of Agriculture. Washington, D. C, May 6, 1905. I *3 * * UNIVERSITY OF FLORIDA 3 1262 08928 3286