NITROGEN IN RELATION TO THE GROWTH OF CITRUS CUTTINGS IN SOLUTION CULTURES A. R. C. HA AS (WITH FIVE FIGURES) The commercial culture of -citrus in most soils of California requires some form of nitrogen fertilization. -Under certain conditions nitrogen may be readily carried to depths below the root zone. In addition, with trees of large size it is frequently difficult to maintain appreciable concentrations of nitrate in the soil solution on account of the large nitrogen requirement of the trees (5). A healthy state of growth in citrus trees frequently is associated with an extremely low concentration of nitrate nitrogen in the soil, and a poor state of growth with a relatively high concentration. In the former case the nitrate is rapidly absorbed while in the latter it is absorbed very slowly because of high concentrations of chlorides or because of some other unfavorable condition. It is desirable, on account of the cost, to keep the nitrate level as low as is consistent with a healthy growth response. In the effort to accomplish this it is possible that the roots during certain periods may be bathed by a soil solution containing little, if any, nitrate or other forms of nitrogen. It has been shown that the growth of citrus in solution cultures is benefited by the temporary depletion of certain elements (3). The length of such depletion period, however, and the nature of the other ions in the solution, determine in a large measure whether or not actual injury shall follow. Deficiency of nitrogen in solution cultures Rooted leafy-twig cuttings of rough lemon were grown in a culture solution of the following composition (p.p.m.) Na K Ca Mg Cl NO3 SO4 P04 B Mn Fe 7 185 159 54 10 718 216 105 0.5 6.2 0.1 Five-gallon capacity shallow enamelware pans were used for the cultures and the solution was not otherwise aerated. It was found by testing the culture solution that nitrogen was the first element to become depleted. The culture solution' was renewed at frequent intervals and the cuttings were grown until they were about four feet high. I In all cultures reported in this paper, phosphate was added for a short time prior to the renewal of the culture solution. When phosphate was absent, 5 p.p.m. of aluminum was added in the form of the citrate to the culture solution. 163
164 PLANT PHYSIOLOGY When this growth was attained, the culture solution was not renewed. After a time, the nitrate became fully depleted while a considerable amount of calcium was still present in the culture solution. This condition was soon followed by a marked response in the appearance of the cuttings. The roots became gelatinous and bluish in color. The tops showed no immediate effect, although there was a gradual loss of leaves during the period of equilibrium restoration between the top and root. The odor of decomposing roots was strong for some distance away whenever a culture vessel cover was momentarily lifted. At this stage, a solution of calcium nitrate was added to the unichanged culture solution sufficient to increase the calcium content by 159 and the nitrate nitrogen by 493 p.p.m. Overnight the odor of the solution disappeared, the colloidal matter in the culture solution settled, and within a few days new growth was evident in the older portion of the root system as seen in figure 1. The gelatinous disintegrating portion gradually separated from the living portion of the root system and settled. Figure 1 A shows the darkening and collapse of the roots after the nitrate nitrogen in the solution was depleted. The effectiveness of adding calcium nitrate to the unchanged culture solution may be. seen in the initial growth of white root tips from the live older portion of the root system. Figure 1 B shows a stage in the later growth of the new roots and illustrates the sloughing away of the gelatinized portions. Control cultures developed excellent roots that did not undergo these changes. In these control cultures the renewal of the solutions was not interrupted exeept in a few cultures in which the occurrence of a period of nitrate deficiency was avooided by the addition of calcium nitrate to the unchanged culture solution. It should be stated, although the causes are not understood, that the growth of citrus cuttings is considerably better in solutions that are frequently changed than in solutions less frequently renewed but in which attempts are made to maintain given concentrations of certain elements. Causes for such a difference may be found to be associated with the concentrations of the so-called minor elements or of toxins in the solution. The effect of a nitrate-nitrogen deficiency on- the roots of leafy-twig cuttings of Lisbon lemon grown in culture solution is seen in figure 1 C. The cuttings were grown for a year without a period of nitrogen deficiency FIG. 1. Nitrogen-deficiency and recovery effects in the roots, of citrus cuttings grown in solution cultures. A, gelatinization of rough lemon roots in, the absence of nitrogen and the initial stage of new root growth following the addition of calcium nitrate to the unchanged nitrogen-depleted culture solution. B, a later stage in the growth of the new roots which is accompanied by the sloughing away of gelatinized roots. - C, similar effects on roots of cuttings of Lisbon lemon in which the period of 'nitrogen deficiency was not prolonged.
HAAS: NITROGEN AND GROWTH OF CITRUS CUTTINGS 165.-r we No Jr I I lkl.. '-W...- t
166-6 -xi PLANT PHYSIOLOGY because of the frequent renewal of the culture solution. Nitrate was then allowed to become deficient in the manner previously described. The period of nitr6gen deficiency was not unduly prolonged before calcium nitrate was added to the unchanged culture solution. New root growth from the living or least injured portion of the root system (fig. 1 C) indicated the beginning of the recovery process. Cuttings of Valencia orange as scion, on cuttings of rough lemon as stock, were grown for a year in a culture solution containing nitrate nitrogen, and then were grown in a culture solution containing no nitrogen. The composition of the nitrate-free' culture solution was as follows (p.p.m.): Na K Ca Mg Cl NO3 SO4 P04 B Mn Fe 7 185 159 54 280 0 216 105 0.5 6.2 0.1 The deficiency was allowed to continue until the growth of the roots was definitely at a standstill before calcium nitrate was added to the solution. The addition of nitrate prevented the collapse of the roots. The deficiency of nitrogen, though not for a period of sufficient duration to destroy the root system, nevertheless severely injured it. The youngest roots failed to respond to the addition of the nitrate. The growth response in the root system during the following year was, therefore, entirely from the older, less injured roots. It was found that small, freshly-rooted cuttings of rough lemon were able to withstand a nitrogen deficiency for a long time when placed directly into the nitrate-free culture solution. The new top growth, if any, was uniformly pale green in color. When cuttings were grown to a considerable size in culture solutions containing nitrate nitrogen and were then grown for a sufficient time in a nitrogen-deficient solution, the roots were injured and the leaves reflected the root condition. The leaves in such cases became pale green, and the veins light colored (fig. 2 A) which usually signifies that the root system is injured. This light-colored vein condition is readily distinguishable from the effects known as chlorosis (fig. 2 B) in citrus leaves in which the veins are green while the remainder of the blade assumes a yellowish green color. Chlorosis may be brought about in many ways, chief among which is the factor of ph. Nitrogen effects on the growth of cuttings Previous investigations (HAAs, 2) have shown that certain forms of nitrogen, when continuously supplied to sand or soil cultures in amounts that insure the absorption of the nitrogen in an unchanged condition, are injurious to citrus, and produce leaf symptoms as evidence of such injury. IJrea, cyanamid, dicyanamid, and closely related forms of nitrogen behave in this way. In view of the fact that little, if any, knowledge exists in
HAAS: NITROGEN AND GROWTH OF CITRUS CUTTINGS 167 FIG. 2. Nitrogen-deficiency effects compared with those of chlorosis in citrus leaves. A, leaf from a cutting of Valencia orange as scion on a eutting of rough lemon as stock. The cutting was grown for a year in a culture solution containing nitrate nitrogen and then in a similar solution lacking nitrogen. B, leaf from a cutting of Valencia orange affected with chlorosis as a result of a high ph value in a culture solution containing nitrate. Note the reverse condition of the veins in A and B. C, leaf from a eutting of Valencia orange grown in a control culture solution. regard to the effect of the form and concentration of nitrogen on the growth of citrus under controlled conditions, preliminary experiments were made in solution cultures as a basis for future investigations. AMMONIA NITROGEN Rooted leafy-twig cuttings of Lisbon lemon were grown from September 19, 1934, to April 25, 1935, in 5-gallon capacity shallow enamelware pans. The culture solution used had the following composition (p.p.m.): Na K Ca Mg Cl S04 P04 B Mn Fe 3.5 71 159 27 275 156 105 0.5 6.2 0.1 The alternation of phosphate with aluminum citrate was carried on as previously mentioned. Ammonium sulphate solution was added to the culture solution in order to secure the following concentrations of nitrogen:
168 PLANT PHYSIOLOGY 0, 1, 2, 3, 5, and 10 p.p.m. The culture solutions were renewed every few days and the ph was maintained at 6 to 6.5 by means of a solution of calcium hydroxide. Growth of the tops was increasingly better as the nitrogen content of the solution increased (fig. 3). The root system was somewhat larger at : v4. FIG. 3. Growth of leafy-twig cuttings of Lisbon lemon in solution cultures to which various concentrations (p.p.m.) of nitrogen were added in the form of ammonium sulphate. the lower than at the higher nitrogen concentrations. In the low nitrogen cultures, some of the leaves showed slight evidence of sulphate excess (HAAs and THOMAS, 4). This is difficult to avoid because of the low nitrogensulphate ratio. Furthermore the absorption of ammonia in the absence of nitrate may not be entirely without injurious effects. In fact, the citrus leaves were yellowish-green in color regardless of the iron supply. The cuttings had the appearance of being in need of nitrogen. These effects of
HAAS: NITROGEN AND GROWTH OF CITRUS CUTTINGS ammonium absorption agree with those obtained in soil cultures that were given daily applications of dilute ammonia. In soil, the discontinuance of the ammonia treatment was followed by recovery as the nitrification of the ammonia progressed. In the present solution cultures, nitrate was not found in the solution at any time, and unless the ammonia was changed to the nitrate form as it was absorbed, the only conclusion possible is that these cultures were grown with enforced ammonia absorption. It is possible, therefore, that a given concentration of ammonium becomes less injurious as the solution or the plant contains increasing amounts of nitrate. Further investigation will be necessary to secure data on the ammonia-nitrate relationship. NITRATE NITROGEN Other cuttings of Lisbon lemon were grown in a culture solution similar to that used for ammonia nitrogen. Nitrogen in the form of potassium nitrate was added to the culture solution in order to obtain the following concentrations (p.p.m.): 0, 1, 2, 3, 5, 10, and 15. Figure 4 shows the resulting growth: that at 3 or 5 p.p.m. was greater than that at 1 and 2 p.p.m. and less than that at higher concentrations. It is doubtful, therefore, whether rapidly growing cuttings, such as lemon, can secure adequate supplies of nitrogen from solutions containing extremely low concentrations of this constituent. In the field, such low concentrations of nitrogen frequently are associated with a healthy vigorous growth in citrus but at some time prior during the year the nitrogen level in the soil far exceeded the low nitrogen concentrations. NITRITE NITROGEN 169 Nitrites have been reported (1) as occurring in considerable concentration in certain soils. The chemical determination of nitrites in soils and plants is a difficult one because of possible changes in the form of the nitrogen that may occur under certain conditions. There has long been a suspicion that nitrite in the field at times may reach concentrations injurious to citrus, but proof of this contention is lacking. Experiments in which solution cultures were used, furnish data regarding the effect of nitrites on citrus. Rooted leafy-twig cuttings of Lisbon lemon were grown from July 23, 1934, to March 22, 1935, in solution cultures in 5-gallon capacity shallow enamelware pans. The culture solution contained (p.p.m.): Na K Ca Mg Cl NO3 S04 P04 B Mn Fe 7 185 159 54 10 785 216 105 0.5 6.2 0.1 The aluminum-phosphate relation was maintained as previously. A solution of potassium nitrite was added to the culture solutions in order to secure
170 PLANT PHYSIOLOGY *:r..~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~......: FIG. 4. Growth of leafy-twig cuttings of Lisbon lemon in solution cultures containing various concentrations (p.p.m.) of nitrogen added in the form of potassium nitrate. the following concentrations of nitrite (p.p.m.) : 0, 5, 10, 15, and 25. The solutions were renewed every few days. The nitrite concentrations were maintained by testing the solutions and adding nitrite as required. The rapidity of change of nitrite to nitrate was retarded by maintaining the ph of the culture solutions in the range 7 to 7.5 by means of a solution of calcium hydroxide. Even so, the nitrite content of the solutions changed considerably within the course of a few days. Figur-e 5 A shows the growth made by the cuttings in solutions containing various concentrations of nitrite. The control cultures were not distinguishablefrom the cultures that received 5 p.p.m. nitrite and hence are not shown in the figure. It is significant that the presence of 785 p.p.m. of nitrate has not prevented the nitrite from seriously interfering with the growth of lemon cuttings. It is not known whether the injury would have been greater in the presence of a smaller concentration of nitrate.
HAAS: NITROGEN AND GROWTH OF CITRUS CUTTINGS 171 FIG. 5. Effect of various concentrations (p.p.m.) of nitrite on the growth of cuttings of citrus in solution cultures. A, Lisbon lemon; B, Valencia orange. In a manner similar to the procedure just described for Lisbon lemon, rooted leafy-twig cuttings of Valeneia orange were grown from October 26, 1934, to April 25, 1935, in solutions containing nitrite. As'shown in figure 5 B the growth of the roots was interfered with when the concentration of nitrite exceeded 5 p.p.m. The cuttings in the'control cultures were not different in appearance from those grown with 5 p.p.m. nitrite. Above 5 p.p.m. nitrite, the finely divided roots were usually dark in color and finally became somewhat gelatinous. Recovery was. rapid when nitrite additions were withheld. The injury brought about by the presence of nitrite is not characterized by symptoms that are more or less specific, hence the diagnosis and the appraisal in the field of the injury for which nitrite is responsible are at present obscure. Summary 1. A preliminary study was made of the growth of citrus cuttings in solution cultures with various forms and concentrations of nitrogen. Such
172 PLANT PHYSIOLOGY a study becomes of importance because the commercial culture of citrus in most soils of California requires some form of nitrogen fertilization. 2. Rooted leafy-twig cuttings of rough lemon or Lisbon lemon were grown in a so-called complete culture solution until the cuttings were several feet high. Depletion of the supply of nitrogen was then followed by a collapse of the root system accompanied by a corresponding loss of affected leaves. The addition of calcium nitrate to the nitrogen-depleted cultures brought about a recovery that varied with the degree of injury. A distinction is shown to occur between the appearance of citrus leaves in need of nitrogen and that due to chlorosis as a consequence of ph. 3. The tops of cuttings of Lisbon lemon grown in solution cultures with ammonium as the only source of nitrogen were larger with increasing concentrations of nitrogen. The leaves had the appearance of being in need of nitrogen. 4. The growth of cuttings of Lisbon lemon was greater with increasing concentrations of nitrate as the source of nitrogen in the culture solution. 5. It is doubtful whether rapidly growing cuttings of citrus can secure adequate supplies of nitrogen from solutions containing extremely low concentrations of this constituent. 6. Coneentrations of nitrite above 5 p.p.m. in culture solutions that contain 785 p.p.m. of nitrate, were distinctly injurious to the growth of cuttings of Lisbon lemon and Valencia orange. UNIVERSITY OF CALIFORNIA CITRUS EXPERIMENT STATION RIVERSIDE, CALIFORNIA LITERATURE CITED 1. FRAPS, G. S. and STERGES, A. J. Occurrence of nitrites in soils. Texas Agr. Exp. Sta. Bull. 412. 1930. 2. HAAS, A. R. C. The zinc relation in mottle-leaf of citrus. Bot. Gaz. 98: 65-86. 1936. 3.. Phosphorus deficiency in citrus. Soil Sci. 42: 93-117. 1936. 4., and THOMAS, E. E. Effect of sulphate on lemon leaves. Bot. Gaz. 86: 345-354. 1928. 5. VAILE, R. S. Fertilizer experiments with citrus trees. California Agr. Exp. Sta. Bull. 345. pp. 465-512. 1922.