SOME EFFECTS OF FERTILIZERS ON THE PRODUCTION OF LUCERNE ROOT NODULES.

New Zealand Journal of Agriculture, Volume XXXVIII, Issue 2, 20 February 1929, Page 103

SOME EFFECTS OF FERTILIZERS ON THE PRODUCTION OF LUCERNE ROOT NODULES.

W.D. Reid,

B.Sc., Mycological Laboratory, Plant Research Station, Palmerston North.

In the establishment of lucerne it is recognized that the formation of root nodules during the growth of the first season is essential for satisfactory results. In some areas in New Zealand artificial inoculation of the seed or soil is not practised — example, in Marlborough, Central Otago,.. and Poverty Bay ; but in other districts, where the conditions for the growth of lucerne are not so favourable, inoculation of the seed by z cultures, or of the area by infected soil, is considered necessary. In all cases, of course, inoculation is required, but in the three districts first mentioned, the grower (perhaps unknowingly) relies upon sufficient inoculation being obtained from . the bacteria already present on the seed or in the soil of the area to be sown down.

For a number of years this Laboratory has been supplying farmers with cultures (on agar slopes) for the inoculation of lucerne-seed, and although the results have been very satisfactory a number of cases have occurred in which nodule - production has been negligible or retarded. These same cultures when used in soil tests at the laboratory have given excellent nodule - formation, and the cause of the failure of the field inoculations required an investigation.' The laboratory experiments were at first restricted to . sowings in soil, the details of farm procedure being followed as closely as possible. Now, in New Zealand it is the custom to mix the seed with the fertilizer prior to sowing, but this fact was not considered in the first experiments. Instead the fertilizersuperphosphate—was sown-in drills and the seed sown upon this manure. The obvious results of these sowings indicated the deleterious effect of superphosphate on the germination of the seed, and it was inferred ■ that a similar effect on the nodule bacteria would be produced. This reduction of germination due to superphosphate is well known to those who have studied the effects of manures, and Mr. A. W. Hudson, Crop Experimentalist, suggested the use of the various manures sown with lucerne-seed, together with one of equal parts of superphosphate and lime, a mixture which had been giving good crop results and little loss in germination in Canterbury.

In order to enable a number of tests to be performed without the delay required for the production of nodules on the plants, plating methods were used,. in which the viable bacteria after treatment were allowed to produce visible colonies on agar plates.* To eliminate possible contaminations of the plates by bacteria adhering to the seed, sterilization of the seed was required. The usual method of sterilization by dipping in corrosive sublimate (1-1,000) was unsatisfactory, since

this treatment prevented the growth of the bacteria of the subsequent inoculation, but of the many disinfectants tested ethyl alcohol was found satisfactory.*

The -following procedure was adopted in the various tests performed : —

(i) A commercial sample of lucerne-seed of good germination was sterilized bv the use of ethyl alcohol, washed in sterile water, and dried.

(2) The inoculation of nodule bacteria was applied to the seed, skim-milk being used for the purpose. Under farming conditions drying does not take more than half an hour, but in the laboratory drying between sheets of sterile absorbent paper occupies about one hour.

(3) The inoculated seed was mixed with the manure to be tested and allowed to remain in contact for half an hour. This period agrees approximately with the time , which would elapse under field conditions, but in some tests where the seed was left in the manure for as short a time as fifteen minutes'the results agreed with those, for the longer periods. Although the quantities of seed and manure were used in the same proportions as for field sowings 14 lb. seed and 3 cwt. manure per acre — this proportion was not essential, as the same result is given by a reduction of the manure to a quantity only sufficient to cover the seed.

(4) After leaving the seed and manure in contact for the desired time, each seed was removed, shaken to remove surplus manure, and then placed upon the surface of the agar-medium plate in the petri dish. Ten seeds were placed on each plate of 3| in. diameter. Alternate seeds were “ wiped ” over the corresponding sectors of the plate, and the plate was then incubated for at least seven days at 25 0 C.

The removal of the surplus manure before plating does not agree with the practice of sowing together the manure and seed on the farm, but such a laboratory method was necessary in order to study the comparative colony growth. In all, fourteen tests were carried out during last year, and a typical result is given in the following table : —

but a study of the number of colonies produced on the “ wiped ” areas (see photos) supplies a better method of comparing the various effects. •

In the case of superphosphate and basic superphosphate the number of colonies which developed on the wiped areas fluctuated between nil and ten colonies for each area, whereas on those plates which received seed which had been treated with slag the number of colonies was indefinitethat is, more than one hundred per area. In these tests the manures were not sterilized, since it was found that,

with the exception of fungi in the super-lime mixture, they were sterile, or were free from any bacteria which could produce colonies similar to those of the nodule bacteria.

As is indicated by the photographs, the number of colonies produced increase as the basic nature of the manure is increased. No attempt is made herein to propound any theory to account for the results which have occurred.

In addition to the plate tests, soil tests were also made, utilizing the correct seed and manure quantities. The results have been equally striking, even after allowing three months’ growth before examination, as was the case with the example shown in the following table : —

Although all the soil tests are not so consistent in results as are the plate tests a fair agreement is present, and they at least confirm the general conclusion derived from the plating method. Such factors as amount of milk film, age and condition of manure, source of manure, origin of culture, have not been considered in detail, since these factors are masked by the application of the manures. Similarly in the soil

tests variations must occur in the conditions of sowing, particularly conditions of moisture content of the soil, and it is recognized that variations — even under so-called controlled conditions — must result in repeated tests. Under field conditions success has been obtained by the use of superphosphate manures, but in these instances the manure has not been mixed prior to sowing, but “ fed ” through a separate box, contact taking place between seed and manure during the passage through the coulters. Since this period of dropping through the coulters is very short it is possible for many of the milk-film protected bacteria to survive.

CONCLUSIONS.

These experiments show that where superphosphate fertilizers are mixed with the inoculated seed before sowing the nodule bacteria are killed; and that where rock phosphate or- basic slag is utilized excellent results are obtained. - B Since this method of applying cultures to the seed is the most satisfactory for New. Zealand conditions, it is advisable to refrain from using the “ acid ” phosphates, or, where such are the only manures available, to sow the seed and manure separately or through separate boxes of the drill.

* Mannite-agar to which 0-5 per cent. CaCo 3 is added. F. Lohnis and N. R. Smith, Jour. Ag. Research, Vol. 6, No. 18, p. 686.

Note. — Supers A and B were obtained from different sources of manufacture. The table gives for each manure the number of seeds which produced colonies of the nodule bacteria. ’ This result in itself is instructive, * A paper dealing with a study of some methods of sterilization of lucerneseed will be published elsewhere.

Fertilizer. Time of Contact of Fertilizer prior to Plating. Number of Seeds sown on Plates. Number of Seeds producing Colonies. Controlno manure and no inoculation • IO Nil Controlno manure plus inoculation IO IO Super A . . . . . • .. i hr. II 4 Super B . . . . ■ . . i hr. II • " ' 5 Basic super . i hr. IO All Super plus lime . . 1 hr. IO All. Basic slag ■ . . . . J hr. IO All

Table 1.

« Fertilizer. Germination on roo Seeds sown per Box. Number of Plants with Nodules. Number of Plants without Nodules. Superphosphate (A) 30 2 28 Superphosphate (B) .. 25 I ' 24 Basic superphosphate 41 2 39 Superphosphate plus lime 53 26 27 Nauru rock phosphate 6 3 50 13 Basic slag .. . . ' 58 52 6

Table 2.