SCHEME I Soil organic'matter
IX. The Essential Plant Micronutrients
A. TERMINOLOGY
T h e r e q u i r e m e n t s b y plants, microorganisms, a n d animals for m i n e r a l elements in small a m o u n t s has been accepted, as likely for n e a r l y as long as t h e subject of m i n e r a l nutrition has been studied.
N u m e r o u s t e r m s t h a t h a v e been used to describe such elements in-clude m i n o r element, trace element, Spurenelement, oligo-element.
Such t e r m s h a v e certain disadvantages because t h e y m a y i m p l y either secondary i m p o r t a n c e or r a r i t y , a n d this m a y be misleading. A r n o n (11a) introduced t h e t e r m m i c r o n u t r i e n t element (abbreviated to m i c r o n u t r i e n t ) . T h i s does not h a v e some of the limitations mentioned a n d is still descriptive. T h e t e r m is n o w w i d e l y used a n d is adopted h e r e a n d in Chapter 2.
B . DISCOVERIES OF ESSENTIAL M I C R O N U T R I E N T S
T h e introduction of n u t r i e n t solutions containing the m i n i m u m n u m -ber of salts w h i c h w o u l d provide t h e elements k n o w n at t h e t i m e to be essential thus p r e p a r e d t h e w a y for m o r e systematic studies on t h e
TABLE XXVI
EXAMPLES OF THE COMPOSITION OF NUTRIENT SOLUTIONS 1. Knop 1865 (134)
gm/1
K N 03 0 . 2
C a ( N 03)2 0 . 8 K H2P 04 0.2 M g S 047 H20 0 . 2
F e P 04 0.1
2. Van der Crone 1904 (258)
gm/1
K N 03 1.0
C a3( P 04)2 0.25
C a S 04 0.25
F e3( P 04)27 H20 0.25 M g S 047 H20 0.25 3. Hoagland and Snyder 1933 (110)
gm/1
KNO3 0.51
C a ( N 03)2 0.82 M g S 047 H20 0.49 K H2P 04 0.136
Iron as 0.5 % iron tartrate at rate of 1 ml per liter when required Supplements A and Β (1 ml/1 of each) containing in 18 liters the weights (grams) shown:
A. (A1)2(S04)3, 1.0; KI, 0.5; KBr, 0.5; T i 02, 1.0; SnCl22H20, 0.5; LiCl, 0.5;
M n C l24 H20 , 7.0; H3B 03, 11.0; Z n S 047 H20 , 1.0; C u S 045 H20 , 1.0;
N i S 046 H20 , 1.0; C o ( N 03)26 H20 , 1.0
B. A s203, 0.1; BaCl2, 0.5; CdCl2, 0.1; B i ( N 03)2, 0.1; R b2S 04, 0.1; K2C r 04, 0.5; KF, 0.1; PbCl2, 0.1; HgCl2, 0.1; M o 03, 0.425; H2S e 04, 0.1; SrS04, 0.5; VC13, 0.1
4. Arnon and Hoagland 1940 (12)
gm/1
K N 03 1.02
C a ( N 03)2 0.492 N H4H2P 04 0.23 M g S 047 H20 0.49 mg/1
H3B 03 2.86
M n C l24 H20 1.81 C u S 045 H20 0.08 Z n S 047 H20 0.22 H2M o 04H20 0.09 F e S 047 H20 0.5% 1
0.6 ml/1 3 X weekly Tartaric acid 0.4 % / 0.6 ml/1 3 X weekly
TABLE XXVI (Continued)
5. Nutrient solution suitable for several coniferous species from sowing to sapling stages. Swan (242). (pH 5.0)
ral/l Parts per million
C a ( N 03)2 3 Ca, 120; Ν, 84
K N 03 2 Κ, 78; Ν, 28
N H 4 C I 2 Cl, 71; Ν, 28
M g S 047 H20 2 Mg, 48; S, 64
N a H2P 042 H20 0.2 Na, 4.6; P, 6.2 Fe as ferric sodium
ethylenediamine
di (O-hydroxypheny lacet ate) 5.0
Mn as MnCl24H20 0.22
Zn as ZnCl2 0.048
Cu as CuCl22H20 0.018
B as H 3 B O 3 0.44
Mo as M0O3 0.033
6. Long Ashton formula, modified from Hewitt (99)
gm/1 ppm mM/1
K N 0 3 0.505 K, 195; N, 70 5
C a ( N 03)3 0.820 Ca, 200; Ν, 140 5
N a H2P 042 H20 0.208 Ρ, 41 1.33
M g S 047 H20 0.369 Mg, 24 3
Ferrie citrate 0.0245 Fe, 5.6 0.1
or at 2.8 ppm (0.05 mM/1) as ferric potassium ethylenediaminetetra acetate prepared as given by Jacobsen (117)
M n S 04 0.00223 Mn, 0.55 0.01
C u S 045 H20 0.000240 Cu, 0.064 0.001
Z n S 047 H20 0.000296 Zn, 0.065 0.001
H3B 03 0.00186 B, 0.37 0.033
( N H4)6M o702 44 H20 0.000035 Mo, 0.019 0.0002
C o S 047 H20 0.000028 Co, 0.006 0.0001
NaCl 0.00585 CI, 3.55 0.1
essential n a t u r e of other elements. T h e r e were, however, n u m e r o u s reports of additional r e q u i r e m e n t s d u r i n g a period of seventy-five years after t h e early w o r k of Salm-Horstmar. T h e first major event in this period was the observation of t h e essential n a t u r e of m a n g a n e s e for Aspergillus niger b y Raulin ( 1 9 4 ) . T h i s w a s confirmed b y B e r t r a n d a n d Javillier (23, 24, 2 5 ) . Javillier (119) showed the importance of zinc for fungi a n d these observations w e r e extended b y B e r t r a n d a n d Javillier ( 2 3 a ) . Confirmation of t h e i m p o r t a n c e of m a n g a n e s e for higher plants represented b y oat (Avena sativa), soybean (Glycine max), pea (Pisum sativum), cowpea (Vigna sp.), tomato a n d other species was obtained b y M c H a r g u e (152, 153). Conclusive experiments
on " g r a y speck" of oats a n d on " m a r s h spot" of peas b y Samuel a n d P i p e r (209, 210) a n d b y P i p e r (186) showed t h a t m a n g a n e s e deficiency is the cause of these disorders.
T h e i m p o r t a n c e of zinc for higher plants represented b y m a i z e (Zea mays), w a s indicated b y M a z e (159) a n d confirmed in a spectacular m a n n e r for several species, including b a r l e y (Hordeum vulgare), sun-flower (Helianthus annuus), b u c k w h e a t (Fagopyrum esculentum), broad b e a n (Vicia faba), k i d n e y b e a n (Phaseolus vulgaris), b y S o m m e r a n d L i p m a n (222) a n d S o m m e r ( 2 2 0 ) . Copper was t h e n e x t element for w h i c h essential requirements w e r e shown in simultaneous a n d inde-p e n d e n t exinde-periments b y S o m m e r (221) a n d b y L i inde-p m a n a n d M c K i n n e y
(148) on barley, flax (Linum usitatissimum), sunflower, a n d tomato.
F u r t h e r w o r k to confirm the general importance of copper for higher plants was carried out b y P i p e r (187) in studies w i t h pea, lucerne
(Medicago sativa), s u b t e r r a n e a n clover (Trifolium subterraneum), tomato, w h e a t (Triticum aestivum), Lolium subulatum, Phalaris tuberosa, a n d flax.
T h e essential n a t u r e of boron w a s first suggested from the work of Aghulon (1) a n d shortly after b y M a z e (160, 161) for maize. T h i s discovery was confirmed beyond a n y doubt for broad bean (Vicia faba) b y t h e thorough work of W a r i n g t o n ( 2 7 2 ) .
T h e importance of m o l y b d e n u m was recognized first for micro-organisms b y Bortels (29, 3 0 ) , w h o found t h a t Azotobacter vinelandii a n d A. chroococcum required m o l y b d e n u m for m a x i m a l rates of nitro-gen fixation. Bortels also showed t h a t m o l y b d e n u m could be partially replaced in these species b y v a n a d i u m w i t h a reduced effectiveness of about 6 0 % of t h a t of m o l y b d e n u m for nitrogen fixation. H o a g l a n d a n d S n y d e r (110) concluded t h a t a n u t r i e n t supplement (A-Z solution) w h i c h provided twenty-two elements in addition to t h e k n o w n nutrients including boron, m a n g a n e s e , zinc, a n d copper, produced additional growth in s t r a w b e r r y (Fragaria) plants in w a t e r culture. L a t e r A r n o n
(9) similarly observed unexplained improvements in growth of barley (Hordeum vulgare) w i t h a m m o n i u m sulfate w h e n given c h r o m i u m , m o l y b d e n u m , a n d nickel in addition to k n o w n nutrients. A r n o n (10) t h e n found t h a t t h e growth of lettuce a n d asparagus (Asparagus of-ficinalis) in w a t e r culture was stimulated b y a group of seven elements w h i c h h e grouped together as A 7 : n a m e l y , m o l y b d e n u m , v a n a d i u m , c h r o m i u m , nickel, cobalt, tungsten, a n d t i t a n i u m . T h e addition of thirteen others (A13) comprising a l u m i n u m , arsenic, c a d m i u m , beryl-lium, fluorine, bromine, iodine, selenium, strontium, lithium, r u b i d i u m , lead, a n d m e r c u r y , produced no further effect.
T h e unequivocal demonstration of t h e essential n a t u r e of m o l y b
-d e n u m for higher plants nevertheless occurre-d unexpecte-dly. Stout a n -d A r n o n (239) h a d recently described improved techniques based on t h e earlier methods of Steinberg (227, 228) for t h e removal of m a n g a n e s e , iron, copper, a n d zinc from stock solutions of n u t r i e n t reagents a n d for the elimination of contamination b y these elements in other c u l t u r e m a -terials. I n some experiments w i t h tomato plants g r o w n b y these methods but given all t h e essential elements t h e n k n o w n , A r n o n a n d Stout (16) observed striking leaf symptoms of mottling a n d necrosis caused b y a physiological disorder not previously recorded. These symptoms w e r e cured w h e n t h e Β7 solution of A r n o n (10) was added. T h e whole effect of this solution could be obtained b y adding one component alone, n a m e l y m o l y b d e n u m at 0.01 p p m , w h e r e u p o n growth w a s rapidly restored to n o r m a l . Although A r n o n a n d Stout (16) attributed t h e dis-covery to the purification m e t h o d adopted, it is not generally accepted
[ H e w i t t ( 9 9 ) , P i p e r ( 1 8 5 ) ] t h a t the methods in question, n a m e l y the precipitation of h e a v y metals w i t h phosphates adsorbed at a n alkaline p H on calcium carbonate a n d phosphate is p a r t i c u l a r l y effective for the removal of m o l y b d e n u m , a n d it is not n o w r e c o m m e n d e d for this p u r -pose. It is likely t h a t t h e discovery of m o l y b d e n u m deficiency in this work was m a i n l y due to t h e use of reagents exceptionally free from this element, a n d in combination w i t h other effective precautions ( 2 3 9 ) , w h i c h excluded its presence from w a t e r a n d containers. T h i s w o r k was closely followed i n d e p e n d e n t l y b y t h a t of P i p e r (185) w i t h Algerian oats. H e w i t t a n d Jones (105) extended t h e investigations to include brassica crops a n d m u s t a r d (Sinapis alba) a n d reproduced for the first t i m e experimentally t h e field disorder of cauliflower w i d e l y a n d long k n o w n as " w h i p t a i l , " w h i c h h a d been described in 1924 b y Clayton ( 5 6 ) . T h e significance of this problem is discussed i n Chapter 2 a n d elsewhere ( 1 0 2 ) . It is interesting to note t h a t "yellow leaf spot" of citrus described b y Floyd in 1908 (75, 76) was identified as long as fifty years later b y Stewart a n d L e o n a r d (232, 233) as a disorder caused b y m o l y b d e n u m deficiency. Both problems are associated with acid soil conditions, b u t whiptail also occurs in n e u t r a l soils w h e n t h e y are severely m o l y b d e n u m deficient.
T h e history of investigations on t h e essential n a t u r e of chlorine is also interesting. About a h u n d r e d years ago, Nobbe a n d Siegert (177, 178) concluded t h a t chlorine w a s r e q u i r e d b y b u c k w h e a t (Fagopyrum esculentum); since t h e n several attempts, n o t a b l y b y Maze (160, 161) a n d b y L i p m a n ( 1 4 7 ) , h a v e produced strong indications t h a t chlorine m i g h t be a n essential element for higher plants.
Although these specific attempts h a d been m a d e to ascertain w h e t h e r plants r e q u i r e chlorine, t h e results w h i c h led to t h e conclusion t h a t it
is i m p o r t a n t for tomato w e r e obtained b y Broyer, Carlton, Johnson, a n d Stout (47) unexpectedly in t h e course of experiments designed to test w h e t h e r cobalt is required. Precautions w e r e directed to eliminating cobalt from water, containers, a n d n u t r i e n t reagents. T h e m e t h o d of n u t r i e n t purification w h i c h was used involved the coprecipitation of cobalt as a sulfide w i t h silver as a collector. T h e silver also combined w i t h halides a n d t h e r e b y removed a substantial p a r t of these impurities.
L a t e r w o r k showed t h a t recrystallization was m o r e effective t h a n pre-cipitation b y silver for removal of chloride a n d bromide from n u t r i e n t reagents. T o m a t o plants g r o w n in these experiments developed a brown-ing a n d necrosis of t h e leaves w h i c h h a d not previously been recorded.
A m a r k e d response was observed in growth w h e n increasing a m o u n t s of cobalt chloride w e r e given, b u t these supplements appeared to exceed in q u a n t i t y w h a t m i g h t be expected to be a d e q u a t e for a micronutrient, w h e n such elaborate precautions w e r e necessary to demonstrate the r e q u i r e m e n t in t h e first place. Reinvestigation of t h e stimulation pro-duced b y some of the unpurified n u t r i e n t reagents a n d b y the chloride anion provided b y cobalt chloride, revealed t h a t t h e deficient element was chlorine a n d not cobalt. F u r t h e r w o r k b y Johnson, Stout, Broyer, a n d Carlton (125) w i t h several crops showed m a r k e d responses w i t h lettuce, lucerne, a n d brassicas. U l r i c h a n d Ohki (256) extended the list to include sugar beet. T h e substitution of chlorine b y b r o m i n e was also shown in t h e work of Johnson et al. (125) a n d of U l r i c h a n d Ohki
( 2 5 6 ) .
Sodium has long been k n o w n to h a v e beneficial effects u p o n higher plants w h e n deficient in potassium, a n d investigations on possible sodium r e q u i r e m e n t s for plants h a v e been n u m e r o u s since the time of Hellriegel a n d W i l l f a r t h ( 9 5 ) . T h i s aspect of sodium nutrition is dis-cussed in Chapter 2. T h e r e is however evidence t h a t sodium m a y be essential for both higher plants a n d microorganisms. Brownell and W o o d (46) concluded t h a t sodium is specifically required in the pres-ence of potassium b y Atriplez vesicaria w h e n g r o w n in w a t e r cultures w i t h special precautions to eliminate sodium. As little as 2.3 p p m ap-peared to be adequate. Allen a n d A r n o n (6) found t h a t sodium was necessary i n addition to potassium for the growth of Anabaena cylindrica.
T h e importance of cobalt has been recognized for microorganisms, especially Lactobacillus lactis (217) for several years in relation to its presence in cyanocobalamin compounds of the B12 group, and m o r e recently in its elemental form or as Bi 2 b y some blue-green algae in-cluding . Nostoc muscorum studied b y H o l m e - H a n s e n , Gerloff, and Skoog ( 1 1 2 ) . H u t n e r , Provasoli, Schatz, a n d Haskins (113) concluded
t h a t Euglena was relatively sensitive in showing a r e q u i r e m e n t for cobalt. T h e question w h e t h e r cobalt is required b y higher plants is u n -decided in spite of t h e careful work of Broyer et al. ( 4 7 ) . S y m p t o m s of a n a p p a r e n t cobalt deficiency w e r e described for cocoa (Theobroma cacao) b y Maskell, Evans, a n d M u r r a y ( 1 5 8 ) . Bolle-Jones a n d Mallikarjuneswara (28) concluded t h a t cobalt stimulated t h e growth of r u b -ber (Hevea brasiliensis) seedlings. M o r e recently A h m e d a n d E v a n s (2, 3, 3 a ) , Reisenauer (196) Delwiche, Johnson, a n d Reisenauer ( 6 5 ) , a n d Halls worth, Wilson, a n d Greenwood (93) i n d e p e n d e n t l y found t h a t cobalt stimulated a n d was clearly required for t h e g r o w t h of nodu-lated soybean (Glycine max) a n d alfalfa (Medicago sativa) a n d sub-t e r r a n e a n clover (Trifolium subsub-terraneum) plansub-ts w h e n dependensub-t on atmospheric nitrogen fixation. T h i s effect m a y reflect a cobalt require-m e n t b y t h e rhizobia or for t h e syrequire-mbiosis r a t h e r t h a n for t h e host p l a n t since A h m e d a n d E v a n s (3a) w e r e able to reduce yields to less t h a n one-tenth t h a t of controls b y omission of cobalt w h e n nitrogen fixation was involved b u t could not observe t h e slightest effect of cobalt w h e n n i t r a t e w a s given i n t h e s a m e experiments w i t h soybean. It is n o t clear w h e t h e r the response to cobalt, w h i c h leads to greatly increased v i t a m i n Bi2 production, is d u e to t h e formation of leghemoglobin; although t h e increase in hemoglobin content of nodules was clearly related to cobalt supply it w a s small in proportion to t h e increased growth a n d nitrogen fixation a n d t h e B12 production t h a t occurred over the same r a n g e of added cobalt. T h e first direct effect of cobalt appeared to be on nitrogen fixation r a t h e r t h a n on the growth of Rhizobium according to Delwiche, Johnson, a n d Reisenauer ( 6 5 ) ; these authors found t h a t t h e fixation process could be detected i n 2 hours i n excised nodules on giving cobalt w h e r e a s growth of Rhizobium w a s not affected u n d e r these conditions.
T h e discovery of A r n o n a n d Wessel (17) t h a t v a n a d i u m is required b y Scenedesmus obliquus was also, as described for chlorine, t h e result of experiments intended to investigate other problems: in this instance, t h e interrelationships between iron supply a n d growth. A r n o n a n d Wessel observed t h a t increasing a m o u n t s of iron, in t h e presence of other k n o w n micronutrients, produced a stimulation in growth w h i c h w a s a p p a r e n t for a m o r e extended r a n g e of iron concentrations t h a n would be expected. T h e major n u t r i e n t reagents w e r e purified b y a sulfide coprecipitation m e t h o d (99, 2 4 0 ) , a n d t h e y w e r e relatively free from v a n a d i u m as well as from m o l y b d e n u m . T h e ferric chloride was not however purified, a n d the presence of a n unidentified m i c r o n u t r i e n t was suspected. W h e n ferric chloride, purified b y extraction w i t h ether in the presence of 6 Ν hydrochloric acid (99, 186, 1 8 7 ) , was substituted for the unpurified compound the growth stimulation was greatly
de-creased. It could, however, be replaced b y v a n a d i u m alone. N o stimula-tion was obtained w i t h nickel or cobalt. T h e o p t i m u m concentrastimula-tion w a s about 20 //.g/liter, w h i c h is probably t w e n t y times t h e o p t i m u m for m o l y b d e n u m .
T h e specific r e q u i r e m e n t for v a n a d i u m b y t h e green alga Scene-desmus obliquus is additional to its a l r e a d y established r e q u i r e m e n t for m o l y b d e n u m . A r n o n a n d Wessel (17) forecast t h a t v a n a d i u m m i g h t be required b y green plants in general, b u t v a n a d i u m has not so far been shown to h a v e a n y essential or even clearly established beneficial effect on higher plants.
Claims h a v e been m a d e since t h e early w o r k b y M a z e (159, 160, 161) t h a t a l u m i n u m , silicon, a n d iodine m a y be essential for higher plants (147, 193, 219, 237, 2 6 1 ) . T h e e a r l y w o r k of S o m m e r (219) was u n u s u a l l y interesting in t h a t millet (Pennisetum sp.) plants raised from seed produced b y plants g r o w n in t h e absence or t h e pres-ence of a l u m i n u m in the previous generation, showed a p e r m a n e n t dif-ferential a n d beneficial effect w h e n sown a n d raised in a single con-tainer to w h i c h n o a l u m i n u m was given. A repetition of this curious ex-p e r i m e n t would b e of interest.
T h e r e are also reports t h a t strontium m a y partially substitute for cal-cium in higher plants (270) as it does in some microorganisms (263) a n d r u b i d i u m can certainly partially replace potassium as clearly shown b y Richards ( 1 9 9 - 2 0 1 ) for barley. Selenium m a y be beneficial
(249) for certain accumulator species such as Astragalus bisulcatus according to Trelease a n d Trelease ( 2 5 0 ) . Other reports of interest indi-cating as yet u n p r o v e d r e q u i r e m e n t s for trace elements include the stimulating effect of nickel on oats, as recorded b y Roach a n d Barclay ( 2 0 2 ) , a n d the occasionally beneficial effects of arsenic, iodine, and other elements noted b y Stiles ( 2 3 6 ) .
C. CRITERIA FOR T H E D E T E R M I N A T I O N OF ESSENTIAL N U T R I E N T REQUIREMENTS
T h e investigation of t h e essential n a t u r e of m i n e r a l n u t r i e n t s involves several problems w i t h respect to both t h e technique a n d t h e standards b y w h i c h essential status m a y be determined. T h i s second point is con-sidered first.
T h e progress of t h e nutritional investigation h e r e reviewed shows t h a t t h e a p p a r e n t l y essential n a t u r e of m a n y elements including those regarded as "trace elements," h a d been discovered a n d accepted b y 1931. T h e logical approach to such studies was, however, p u t on a firm basis b y A r n o n a n d Stout ( 1 5 ) , A r n o n (11) b y t h e introduction of
criteria of essentiality b y w h i c h the status of a n element m i g h t be judged.
T h e s e criteria w e r e outlined as follows: (a) T h e element m u s t be es-sential for n o r m a l growth, or reproduction, w h i c h cannot proceed in its absence, (b) T h e r e q u i r e m e n t for t h e element m u s t be specific a n d not replaceable b y another, (c) T h e r e q u i r e m e n t m u s t be direct, not a m a n i -festation of indirect effects such as antagonism of a toxic effect.
A t t h e t i m e of their proposal these criteria seemed a d e q u a t e a n d u n -ambiguous, b u t since t h e n it h a s become necessary to take account of some other ideas w h i c h m a y sometimes qualify their application.
T h e first criterion would a p p e a r unequivocal. If a p p a r e n t l y stimula-t o r y effecstimula-ts of cerstimula-tain elemenstimula-ts occur astimula-t low levels, b u stimula-t no a b n o r m a l i stimula-t y or serious restriction of g r o w t h can be detected w h e n attempts are m a d e to exclude all traces of such elements, the exact interpretation or appli-cation of t h e first criterion becomes problematic. T h e question is con-sidered below in relation to t h e "beneficial" elements.
It is clear t h a t v a n a d i u m can replace m o l y b d e n u m at comparable or decreased efficiency in certain species of Azotobacter [Burk ( 5 2 ) , Bortels ( 2 9 , 3 0 ) ] or in Clostridium butyricum [Jensen a n d Spencer
( 1 2 3 ) ] . Similarly strontium can replace calcium in Azotobacter [Burk a n d L i n e w e a v e r ( 5 3 ) ] or in Chlorella pyrenoidosa [ W a l k e r ( 2 6 3 ) ] . It follows t h a t if r e p l a c e m e n t is complete, essential r e q u i r e m e n t s for either one of t h e pair cannot be demonstrated if the other element is present, a n d i n t h e absence of both, either can be shown to be ap-p a r e n t l y essential.
It is conceivable t h a t if a n element is required for a single specific role w h i c h can be m a d e superflous b y altering some other aspect of n u -trition, e.g., nitrogen supply or possibly a change from autotrophic to partially heterotrophic nutrition, the particular element m i g h t no longer be essential. T h i s situation appears to occur w i t h t h e molyb-d e n u m r e q u i r e m e n t b y Scenemolyb-desmus obliquus, w h e n nitrate is re-placed b y u r e a or a m m o n i u m carbonate according to v e r y careful work b y Ichioka a n d A r n o n ( 1 1 4 ) . T h e question of essentiality of molyb-d e n u m for other organisms u n molyb-d e r similar circumstances is molyb-discussemolyb-d in Chapter 2 a n d elsewhere ( 1 0 2 ) .
T h e presence or absence of certain elements m a y influence the course of metabolism so t h a t tissue composition or e n z y m e activity are m a t e r i a l l y altered. P l a n t s , however, a p p e a r to tolerate large differences in their composition w i t h respect to certain major metabolic constit-u e n t s inclconstit-uding a m i n o acids, sconstit-ugars, phosphate esters, a n d organic acids, a n d t h e y m a y yield widely differing e n z y m e activities in vitro w i t h n o r m a l external appearance. Such a n element would not thereby
qualify as essential in spite of its capacity to influence metabolism. T h e formation of selenomethionine u n d e r conditions of selenium nutrition m i g h t provide a n example of such a relationship.
T h e problem of a p p a r e n t l y beneficial elements discussed at length in Chapter 2 m u s t b e considered. It is well k n o w n t h a t some reports of beneficial effects of a n element w e r e d u e to failure to decrease t h e threshold levels of t h e element to a point w h e r e severe deficiency effects w e r e produced. It is conceivable, however, as outlined i n C h a p t e r 2, t h a t one element ( A ) m a y replace another (B) at greater efficiency in a certain role (bi) b u t m a y be u n a b l e to fulfill some other function
(bii) for w h i c h Β is essential a n d is not replaced b y A. E l e m e n t A would t h e n fulfill a beneficial b u t n o t essential role. T h e quantitative relationships would d e t e r m i n e w h e t h e r the effect was b a r e l y perceptible or w a s t a n t a m o u n t to a quasi-essential function. Beneficial elements include also those w h i c h m a y partially substitute for a n essential ele-m e n t u n d e r deficiency conditions. Sodiuele-m a n d potassiuele-m coele-mprise the best-known example of this relationship, as discussed in Chapter 2.
T h e question w h e t h e r a n element is essential cannot be answered in a negative sense. Stout a n d A r n o n ( 2 3 9 ) , w h o developed greatly im-proved methods for the s t u d y of n u t r i e n t r e q u i r e m e n t s of plants, emphasized t h a t w h e n attempts to show a r e q u i r e m e n t for a particular element yielded negative results it was possible o n l y to say that, if required, t h e necessary concentration of t h e element w a s below t h e limits of t h e methods used to eliminate t h a t e l e m e n t or to detect its presence. It is, however, equally i m p o r t a n t to recognize t h e possibility t h a t not all t h e chemical elements in t h e periodic table are in fact es-sential to all, or even to a n y , living cells a n d t h e negative results m a y reflect a nonessential character even though this cannot be proved. T h e r e q u i r e m e n t s of various organisms a r e clearly diverse, a n d it is not justifiable at present to argue from t h e particular to the general b y suggesting t h a t t h e observation of essential r e q u i r e m e n t s for a certain element b y one organism, e.g., for v a n a d i u m b y Scenedesmus ( 1 7 ) , m a k e a r e q u i r e m e n t likely for green plants as a group, as A r n o n a n d Wessel suggest. T h e r e q u i r e m e n t for individual elements m a y differ qualitatively for different organisms a n d w h a t is essential for one m a y not be required b y another, p a r t i c u l a r l y as evolution of protein speci-ficity m a y change the extent to w h i c h a particular element can serve as a prosthetic group.
I n spite of t h e earlier emphasis on t h e difficulty of interpreting nega-tive results, i.e., of proving t h a t a n element is not required, Ichioka a n d A r n o n (114) definitely concluded t h a t for Scenedesmus, m o l y b d e n u m was not required at all w h e n t h e alga w a s g r o w n w i t h u r e a or a m
-m o n i u -m carbonate. Critical inspection of t h e i r data does i n fact show no response whatsoever to m o l y b d e n u m u n d e r these conditions; if it is required, t h e concentration m u s t be t w o or t h r e e orders lower t h a n concentrations t h a t produce detectable effects w i t h all other organisms t h a t h a v e been tested u n d e r similar conditions of nitrogen supply.
A r n o n a n d associates ( 1 3 ) concluded that, i n t h e presence of n i t r a t e , the m o l y b d e n u m level w a s 1 . 5 X 1 03 atoms p e r cell w h e n deficiency effects w e r e seen. T a k i n g into consideration t h e experience gained from a great m a n y experiments w i t h different organisms b y several workers a n d from prolonged studies w i t h h i g h e r plants a t L o n g Ashton as described i n Chapter 2 , it m a y b e concluded t h a t if m o l y b d e n u m is required b y Scenedesmus i n t h e presence of u r e a or a m m o n i u m carbonate, t h e level at w h i c h a deficiency would be observed m i g h t well be o n l y about 1 5 atoms p e r cell. T h e question is therefore still open, b u t t h e conclusion of Ishioka a n d A r n o n ( 1 1 4 ) appears reasonable a n d m a y b e correct.
X. Experimental Methods for the Study of Micronutrient Requirements