the effect of three factorial levels of nitrogen of

THE EFFECT OF THREE FACTORIAL LEVELS OF NITROGENAND PHOSPHORUS ON THE GROWTH AND COMPOSITION

OF CINCHONA LEDGERIANA

AR N AU D J. Lou STAL O T AND HA R O L D F. WINTER S

(WITH ONE FIGURE)

Received October 27, 1947

Most of the cultural trials and experiments with cinchona prior to 1900were recorded as observations. AWith the possible exception of the workby COWGILL (1) the published information with reference to the effects offertilizer applications onl growth and alkaloid content of cinchona dealmainly with established trees (3, 4, 8, 9, 11). Although it is generally con-ceded that the first few months are the most critical period in the life of thecinchona plant, little or no experimental data are available as to the nutri-tional requirements of young seedlings.

Nitrogen and phosphorus are two essential mineral elements that areusually deficient in tropical soils. In view of this fact an experiment wasinitiated to obtain information on the effects of three levels of each of thoseelements and their inter-relationship on the growth and composition of youngeinchona seedlings.

Materials and methods

Seedlings of Cinchona ledgeriana selected for uniformity were trans-planted in July 1946 to 5-gallon crocks filled with medium coarse silica sand.Five seedlings were planted to each crock. One replication which consistedof nine crocks was placed in each of three air-conditioned greenhousechambers described elsewhere (10). The temperature in all chambers wasmaintainied at 750 F. during the day and 650 F. at night. In a previousexperiment (10), this temperature was found to be the most favorable forgrowth of C. ledgeriana. Relative humidity was maintained as high aspossible by continually spraying the floors with water. During the middleof the day relative humidity usually dropped to 60 or 707% but most of thetime it ranged betweeni 80 and 95%.

During July and August the plants in all crocks were supplied withHoagland and Arnon's complete nutrient solution (5) diluted to 1/4 strengthand adjusted to pH 5.5. One gallon of solution was applied to each crockby pourinig on the surface of the sand three or four times a week. A three-quarter-inch hole plugged with glass wool at the bottom of each crock pro-vided adequate drainage.

In September, two or three of the weakest plants in each pot were re-moved and the differential nutrient solution treatments started. These con-sisted of three levels of nitrogen and three levels of phosphorus in factorialcombination. The low, medium, and high levels of nitrogen were supplied at

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3, 18, and 81 p.p.m. and the low, medium, and high levels of phosphorus weresupplied at 0, 5, and 25 p.p.m. respectively. The other essential mineralelements were maintained at equal levels in all solutions. The compositionof the nine factorial nutrient solutions are given in table I. Each nutrientsolution was made up in 50-gallon drums, adjusted to pH 5.5 and applied asdescribed previously. The position of the crocks within each chamber orreplication was randomized.

After 5 months of treatment the plants were harvested and growth datawhich included height, fresh and dry weight, and top-root ratio wereobtained. The roots and stems were analyzed for total alkaloid and quininesulfate content by the method described by LOUSTALOT and PAGAN (6). Theleaves were analyzed for nitrogen, phosphorus, potassium, calcium, andmagnesium by the methods outlined by DROSDOFF and PAINTER (2).

ResultsEFFECT OF TREXTMENTS ON GROWTH

The first differences due to treatment were evident in the plants of lownitrogen treatment. About one month after the treatments were startedthe leaves of those plants were lighter green in color than those of plants inthe higher nitrogen treatments and there was a definite check in growth. Thechlorosis became more intensified with time. The higher levels of phos-phorus in the N1 group seemed to intensify the effects of low nitrogen. Theplants in those treatments appeared more chlorotic than those of the N,P1,and, in addition, had considerable red coloration in the young leaves, stems,and petioles. This was more pronounced in the N1P3 plants than in theN1P2 plants. The leaves of plants in the N2 series in general were inter-mediate in color between those of the N1 and N? groups. The effect of highphosphorus in the N2 group was similar to that in the N1 series but to alesser degree. The plants in the high phosphorus treatment of the N2 levelwere considerably less green and had more red coloration than those of theP1 and P2 plants at the same nitrogen level.

The seedlings grown at the high nitrogen level (N3) had large dark-greenleaves and thick stems and there was no reddening as in the N1 and N2 groupsregardless of phosphorus level. The plants in the low phosphorus treat-ment had narrow and darker colored leaves than those of plants in the P2and P, levels. However, there were no other clear-cut symptoms that couldbe attributed to phosphorus deficiency.

The growth data presented in table II show that the low level of nitrogenhad a profound depressing effect on the growth of plants in this treatment.The height and fresh and dry weights of seedlings receiving the low levelof nitrogen were significantly lower than those of plants grown at themedium and high levels of nitrogen, but there was no statistically significantdifference between the growth of the N2 and N3 plants.

The high level of phosphorus had a depressing effect on growth in the lownitrogen plants and to a lesser extent in the medium nitrogen group. In the

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345LOUSTALOT AND WINTERS: GROWTH OF CINCHONA

TABLE ICOMPOSITION OF NITROGEN-PHOSPHORUS NUTRIENT SOLUTIONS*

TREATMENT No. NITROGEN AND PHOSPHORUS NITROGEN PHOSPHORUSLEVELS

p.p.M. p.p.m.1 P1 3 02 N1 P2 3 53 P3 3 254 P1 18 05 N2 P2 18 56 P3 18 257 P1 81 08 N3 P2 81 59 P3 81 25

* The concentration of other essential elements in all solutions were as follows:K-40 p.p.m.; Ca-232 p.p.m.; Mg- 48 p.p.m.; S-48 p.p.m.; Fe 3 p.p.m.; Mn-0.5p.p.m.; B-0.5 p.p.m.; Zn-0.5 p.p.m.; and Cu-0.1 p.p.m.

high nitrogen series the reverse was true. The least growth of the N3 plantswas made when phosphorus was low and vice versa. These results are inagreement with those of COWGILL (1). He conducted an N, P, K, factorialexperiment with Cinchowa ledgeriana in sand culture and also concludedthat phosphorus is required in relatively small amounts by young cinchonaseedlings and that growth may be limited under certain conditions if thephosphorus concentration is too high. He states further that although therelative requirement for phosphorus is low, it must be available in the properproportion to nitrogen and possibly potassium.

The percentage of dry matter in the low nitrogen plants was significantlyhigher than that in the medium and high nitrogen plants. This is notsurprising since the growth of the N1 plants was retarded as a result of nitro-gen deficiency. The top-root ratio of the low and medium ilitrogen seedlings

TABLE IIGROWTH OF Cinchona ledgeriana UNDER THREE FACTORIAL LEVELS OF

NITROGEN AND PHOSPHORUS

TREATMENT NITROGEN AND AEGE AVERAGE AVERAGE DRY TOP-ROOTT No. PHOSPHORUS HEIGHT* FRESH DRY DRLEVELS HGH WEIGHT WEIGHTRAI

cm. gm. gm. %1 P1 48.4 52.1 11.6 25.4 1.042 N1 P2 53.7 66.5 15.7 23.6 1.003 P3 45.8 35.0 8.4 24.0 1.034 P1 71.4 120.0 26.5 22.1 1.005 N2 P2 75.1 125.3 26.7 21.3 1.076 P3 70.1 104.0 23.9 22.9 0.837 P1 61.1 83.0 18.3 22.0 1.378 N3 P2 85.1 128.3 26.9 21.0 2.309 P, 76.2 136.8 28.5 20.8 1.60

* Each figure is an average of 6 to 9 plants.



PLANT PHYSIOLOGY346

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LOUSTALOT AND WINTERS: GROWTH OF CINCHONA

was significantly lower than that of the plants in the high nitrogen treat-ment. Photographs of representative plants from all treatments are shownin figure 1.

EFFECT OF TREATMENTS ON TOTAL ALKALOID AND QUININE SULFATE CONTENTThe total alkaloid and quinine sulfate content of the roots and stems are

given in table III. In general the roots and stems of plants grown at thehigh nitrogen level contained significantly higher amounts of total alkaloidand quinine sulfate than did those of plants in the medium and low nitrogentreatment. There was no statistically significant difference in the amountsof these constituents between roots and stems of the N1 and N2 plants. How-ever, there was a tendency for them to be higher in tissues of the mediumnitrogen plants than in those of the low nitrogen seedlings. There was no

consistent effect of phosphorus on the quinine sulfate content of the plants inthe various treatments, but there was a definite tendency for the total alka-loids to be higher in the plants grown at a high phosphorus level than in thosegrown at a low phosphorus level.

TABLE IIITOTAL ALKALOID AND QUININE SULFATE CONTENT OF Cinchona ledgeriana SEEDLINGS

GROWN UNDER THREE FACTORIAL LEVELS OF NITROGEN AND PHOSPHORUS*

TREATMENT NITROGEN AND TOTAL ALKALOIDS QUININE SULFATENo. PHOSPHORUS

LEVELS STEMS ROOTS STEMS ROOTS

1 P1 2.93 3.05 0.46 0.812 N1 P2 2.99 3.57 0.37 1.063 P3 3.91 3.01 0.43 1.174 P1 3.65 3.91 0.50 1.215 N2 P2 3.22 3.64 0.42 1.226 P3 3.76 4.28 0.46 1.007 P1 3.79 3.72 0.68 1.408 N, P2 4.22 4.25 0.69 1.279 Ps 4.23 4.43 0.54 1.43

* The authors are indebted to Caleb Pagan for making these analyses.

EFFECTS OF TREATMENTS ON MINERAL COMPOSITION OF LEAVES

It is evident from the data presented in table IV that the treatmentshad a marked effect on the mineral composition of the leaves. As wouldbe expected the nitrogen content of the seedlings was directly correlatedwith the level of nitrogen at which they were grown, i.e., the low nitrogenplants contained the least amounts of the element, the high nitrogen hadthe highest, and the medium nitrogen were intermediate in nitrogen con-tent. There was a consistent effect of the phosphorus levels on the nitrogencontent of the leaves. At all nitrogen levels, the high phosphorus plantscontained less nitrogen than did the low phosphorus plants. These dataindicate that applications of phosphorus should not be made to young

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cinchona trees when nitrogen supply tends to be low because the additionof phosphorus might intensify the nitrogen deficiency. This was evidentfrom the retarded growth of the plants in the high phosphorus low nitrogentreatment.

The potassium content of the leaves was not appreciably affected by thenitrogen levels, although there was a tendency for potassium to be somewhathigher in leaves of the N3 plants than in those of the N1 and N2 seedlings.The potassium content of low and medium nitrogen plants at the highphosphorus level was considerably less than similar plants grown in lowphosphorus treatments. At the N3 level phosphorus had no appreciableeffect on the potassium content of the leaves.

The calcium content of the leaves from plants grown in high nitrogensolution was consistently higher than that of leaves from medium and low

TABLE IVMINERAL COMPOSITION OF LEAVES OF Cinchona ledgeriana GROWN UNDER THREE

FACTORIAL LEVELS OF NITROGEN AND PHOSPHORUS

TREATMENT NITROGEN AND PHOS- POTAS- GCALCIUMMG-No. PHOSPHORs No PHORUSS%UM NESIUM

LEVELS

1 P1 1.28 0.24 1.14 0.43 0.292 N1 P2 1.13 0.28 1.14 0.53 0.263 P3 1.10 0.49 0.82 0.67 0.21

4 P1 1.70 0.20 1.21 0.45 0.285 N2 P2 1.45 0.25 1.07 0.57 0.246 P2 1.28 0.37 0.83 0.63 0.22

7 P1 2.45 0.20 1.13 0.75 0.288 N3 P2 2.30 0.21 1.15 0.72 0.279 P2 2.19 0.24 1.19 0.70 0.25

nitrogen plants. There was no marked effect of phosphorus on calcium con-tent of the N3 level but at the N2 and N1 levels of nitrogen the amount ofcalcium in the leaves was directly correlated with the level of phosphorus.

The nitrogen levels had no appreciable effect on the magnesium contentof the leaves. However, at all nitrogn levels there was a small but consistenteffect of phosphorus on the magnesium content. Magnesium was slightlylower at the high phosphorus level.

DiscussionThe results of this experiment, insofar as they are applicable to field

conditions, indicate that young cinchona trees are particularly sensitive tothe fertilizer balance. Careful consideration should be given to all kinds andamounts of fertilizer and the ratios of the fertilizer elements applied. Fromthe data in this paper this is especially true for nitrogen and phosphorus.Nitrogen supply to cinchona seedlings is important both from the standpointof better growth and higher content of quinine and other alkaloids. Ofspecial interest is the fact that supplying 18 p.p.m. of nitrogen to the seed-

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LOUSTALOT AND WINTERS: GROWTH OF CINCHONA

lings resulted in almost as good growth as 81 p.p.m., indicating that nitrogenconcentrations much above this level are not warranted.

Of fundamental interest is the effect of nitrogen and phosphorus levelson mineral composition of the leaves. Leaves of plants supplied a high levelof nitrogen had a fairly uniform composition regardless of phosphorus levelwith the exception of the nitrogen content which varied inversely with thephosphorus supply. However, in plants where nitrogen level was medium orlow the phosphorus level had a definite effect on the mineral composition ofthe leaves. In these plants the percentages of calcium and phosphorus in-creased as the phosphorus supply increased but the amounts of nitrogen,potassium and magnesium decreased. These results are additional evidencein support of the nutrient-element balance concept proposed by SHEAR,CRANE, and MYERS (7). These authors state "as any element decreases orincreases substantially from its concentration of optimum intensity, themaximum growth possible within the new limits of supply of that elementcan result only when the concentration of all elements have been brought intobalance at the new level of intensity determined by that element." In-creasing the level of one element (as phosphorus, in the present experiment)while maintaining another element (nitrogen) at a deficient or low levelresulted in an unbalanced condition in regard to other elements even thoughthey were available in all treatments in equal concentration.

Summary

1. Cinchona ledgeriana seedlings were grown in sand culture at threelevels of nitrogen (3, 18, and 81 p.p.m.) and three levels of phosphorus (0, 5,and 25 p.p.m.) in factorial combination.

2. The low level of nitrogen had a marked depressing effect on the growthof the seedlings, but there was no statistically significant difference in thegrowth of plants receiving 18 and 81 p.p.m. of nitrogen.

3. The high level of phosphorus greatly depressed growth of plants withthe low nitrogen supply and to a lesser extent those supplied with a mediumnitrogen level. On the other hand growth of the high nitrogen plants wasdirectly correlated with phosphorus level.

4. Roots and stems of plants grown at the high nitrogen level containedhigher amounts of total alkaloid and quinine sulfate than did those of plantsgrown at the lower nitrogen levels.

5. There was no consistent effect of phosphorus on the quinine contentof the plants in the various treatments but there was a tendency for totalalkaloids to be higher in plants with a high phosphorus level.

6. The nitrogen content of the leaves varied directly with the nitrogenlevel at which the plants were grown. At all nitrogen levels the nitrogencontent of the leaves was inversely correlated with the phosphorus supply.Thus, at the low nitrogen level the high phosphorus concentration accentu-ated nitrogen deficiency.

7. The leaves of plants grown in the low and medium nitrogen levels

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contained increased percentages of calcium and phosphorus as the phos-phorus supply increased but the amounts of nitrogen, potassium, and magnes-ium, in these plants decreased.

8. The results of this experiment indicate that growth of young cinchonatrees may be limited under certain conditions if the phosphorus concentrationis too high. They also indicate that the phosphorus requirement of cinchonais relatively low and that for optimum growth the phosphorus must beavailable in the proper proportion to nitrogen and possibly other mineralelements.

OFFICE OF EXPERIMENT STATIONSU. S. DEPARTMENT OF AGRICULTURE

MAYAGUEZ, PUERTO RIco

LITERATURE CITED1. COWGILL, W. H. Studies on the germination, early seedling growth

and nutrition of cinchona. Thesis for Ph.D. University of Mary-land. 1944.

2. DROSDOFF, M., and PAINTER, J. H. A chlorosis and necrosis of tungleaves associated with potassium content. Proc. Amer. Soc. Hort.Sci. 41: 45-51. 1942.

3. GOEPFERT, F. The fertilizing of cinchona groves. De BergeulturesIII (3): 68-70. 1928.

4. GROOTHOFF, ARNOLD. Rational management of cinchona plantationsin connection with the factors which influence the quantity andquality of cinchona bark. H. D. Tjeenk Willink & Zoon, Haarlem.94 pp. 1919.

5. HOAGLAND, D. R., and ARNON, D. I. The water-culture method forgrowing plants without soil. Univ. of California Cir. 347. 1938.

6. LOUSTALOT, A. J., and PAGAN, CALEB. A quick and simple method fordetermining quinine and total alkaloids in cinchona bark. Assoc.Off. Agr. Chem. Jour. 30(1): 153-159. 1947.

7. SHEAR, C. B., CRANE, H. L., and MYERS, A. T. Nutrient-element bal-ance: a fundamental concept in plant nutrition. Proc. Amer. Soc.Hort. Sci. 47: 239-248. 1946.

8. SPRUIT, C. Pruning and thinning in regularly fertilized cinchonagroves. De Bergeultures IX(27): 603-610. 1935.

9. SPRUIT, C. Field tests to improve cinchona culture. De BergeulturesIII (57): 1453-1457. 1929.

10. WINTERS, H. F., LOUSTALOT, A. J., and CHILDERS, N. F. Influence oftemperature on growth and alkaloid content of cinchona seedlings.Plant Physiol. 22: 42-50. 1947.

11. VAN LEERSUM, P. The fertilizing of cinchona. Teysmannia, Batavia.20(1): 17-36. 1909.

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