13 Molybdenum Russell L. Hamlin Coggins Farms and Produce, Lake Park, Georgia CONTENTS 13.1 Historical Information 375 13.1.1 Determination of Essentiality 375 13.1.2 Function in Plants 376 13.1.2.1 Nitrogenase 376 13.1.2.2 Nitrate Reductase 377 13.1.2.3 Xanthine Dehydrogenase 377 13.1.2.4 Aldehyde Oxidase 378 13.1.2.5 Sulfite Oxidase 378 13.2 Diagnosis of Molybdenum Status of Plants 378 13.2.1 Deficiency 378 13.2.2 Excess 379 13.2.3 Molybdenum Concentration and Distribution in Plants 379 13.2.4 Analytical Techniques for the Determination of Molybdenum in Plants 382 13.3 Assessment of Molybdenum Status of Soils 382 13.3.1 Soil Molybdenum Content 382 13.3.2 Forms of Molybdenum in Soils 384 13.3.3 Interactions with Phosphorus and Sulfur 385 13.3.4 Soil Analysis 386 13.3.4.1 Determination of Total Molybdenum in Soil 386 13.3.4.2 Determination of Available Molybdenum in Soil 386 13.4 Molybdenum Fertilizers 387 13.4.1 Methods of Application 387 13.4.1.1 Soil Applications 387 13.4.1.2 Foliar Fertilization 388 13.4.1.3 Seed Treatment 388 13.4.2 Crop Response to Applied Molybdenum 388 References 389 13.1 HISTORICAL INFORMATION 13.1.1 D ETERMINATION OF ESSENTIALITY Molybdenum was discovered in 1778 by the Swedish chemist, Carl Wilhelm Scheele. However, its importance in biological systems was not established until 1930 when Bortels discovered that molyb- denum was essential for the growth of Azotobacter bacteria in a nutrient medium (1). Subsequently 375 CRC_DK2972_Ch013.qxd 6/6/2006 1:13 PM Page 375 in 1936, Steinberg determined that molybdenum was required for the growth of the fungus Aspergillus niger (2). The essential nature of molybdenum for higher plants was first reported by Arnon and Stout in 1939 (3). In earlier experiments, Arnon observed that minute amounts of molybdenum improved the growth of plants in solution culture (4), and that a group of seven heavy metals, including molybdenum, increased the growth of lettuce (Lactuca sativa L.) and asparagus (Asparagus officinalis L.) (5). Prior to these studies (conducted in 1937 and 1938, respectively) only boron, cop- per, iron, manganese, and zinc were considered to be micronutrients. The observation that plant growth was improved by elements other than these led Arnon to believe that the list of essential ele- ments was incomplete, and prompted him to test whether or not molybdenum was essential for the growth of higher plants (3). In their studies, Arnon and Stout tested the molybdenum requirement of tomato (Lycopersicon esculentum Mill.) by their newly established criteria for essentiality (6). These criteria were (a) a deficiency of the essential element prevents plants from completing their life cycles; (b) the requirement is specific to the element, the deficiency of which cannot be prevented by any other element; and (c) the element is involved directly in the nutrition of plants. Plants grown in purified solution cultures developed deficiency symptoms in the absence of molybdenum, and symptoms were prevented by adding the equivalent of 0.01 mg Mo L Ϫ1 to the root medium (6). Normal growth was restored to deficient plants if molybdenum was applied to the foliage, thereby estab- lishing that molybdenum exerted its effect directly on growth and not indirectly by affecting the root environment. 13.1.2 FUNCTION IN PLANTS The transition element molybdenum is essential for most organisms and occurs in more than 60 enzymes catalyzing diverse oxidation–reduction reactions (7,8). Although the element is capable of existing in oxidation states from 0 to VI, only the higher oxidation states of IV, V, and VI are impor- tant in biological systems. The functions of molybdenum in plants and other organisms are related to the valence changes that it undergoes as a metallic component of enzymes (9). With the exception of bacterial nitrogenase, molybdenum-containing enzymes in almost all organisms share a similar molybdopterin compound at their catalytic sites (7,8). This pterin is a molybdenum cofactor (Moco) that is responsible for the correct anchoring and positioning of the molybdenum center within the enzyme so that molybdenum can interact with other components of the electron-transport chain in which the enzyme participates (7). Molybdenum itself is thought to be biologically inactive until complexed with the cofactor, Moco. Several molybdoenzymes including nitrogenase, nitrate reductase, xanthine dehydrogenase, aldehyde oxidase, and possibly sulfite oxidase are of significance to plants. Because of its involve- ment in the processes of N 2 fixation, nitrate reduction, and the transport of nitrogen compounds in plants, molybdenum plays a crucial role in nitrogen metabolism of plants (10). 13.1.2.1 Nitrogenase The observation of Bortels (1) that molybdenum was necessary for the growth of Azotobacter was the first indication that molybdenum played a role in biological processes. It is now well established that molybdenum is required for biological N 2 fixation, an activity that is facilitated by the molyb- denum-containing enzyme nitrogenase. Several types of asymbiotic bacteria, such as Azotobacter, Rhodospirillum, and Klebsiella, are able to fix atmospheric N 2 , but of particular importance to agri- culture is the symbiotic relationship between Rhizobium and leguminous crops (10). Nitrogenases from different organisms are similar in nature, and they catalyze the reduction of molecular nitro- gen (N 2 ) to ammonia (NH 3 ) in the following reaction (11): N 2 ϩ8H ϩ ϩ8e Ϫ ϩ16ATP → 2NH 3 ϩH 2 ϩ16ADPϩ16Pi 376 Handbook of Plant Nutrition CRC_DK2972_Ch013.qxd 6/6/2006 1:13 PM Page 376 One of the great wonders in nature is how the process of N 2 fixation takes place biologically at nor- mal temperatures and atmospheric pressure (12), when in the Haber–Bosch process, the same reac- tion performed chemically requires temperatures of 300 to 500°C and pressures of Ͼ300 atm (13). According to Mishra et al. (11), nearly all nitrogenases contain the same two proteins, both of which are inactivated irreversibly in the presence of oxygen: an Mo–Fe protein (MW 200,000) and an Fe protein (MW 50,000 to 65,000). The Mo–Fe protein contains two atoms of molybdenum and has oxidation–reduction centers of two distinct types: two iron–molybdenum cofactors called FeMoco and four Fe-S (4Fe-4S) centers. The Fe–Mo cofactor (FeMoco) of nitrogenase constitutes the active site of the molybdenum-containing nitrogenase protein in N 2 -fixing organisms (14). The effect of biological N 2 fixation on the global nitrogen cycle is substantial, with terrestrial nitrogen inputs in the range of 139 to 170 ×10 6 tons of nitrogen per year (15). Despite the impor- tance of molybdenum to N 2 -fixing organisms and the nitrogen cycle, the essential nature of molyb- denum for plants is not based on its role in N 2 fixation. The primary breach of the Arnon and Stout criteria of essentiality (6) is that many plants lack the ability to fix atmospheric N 2 and therefore do not require molybdenum for the activity of nitrogenase. In addition, the process of N 2 fixation is not essential for the growth of legumes if sufficient levels of nitrogen fertilizers are supplied (11,16). 13.1.2.2 Nitrate Reductase The essential nature of molybdenum as a plant nutrient is based solely on its role in the NO 3 Ϫ reduc- tion process via nitrate reductase. This enzyme occurs in most plant species as well as in fungi and bacteria (12), and is the principal molybdenum protein of vegetative plant tissues (17). However, the requirement of molybdenum for nitrogenase activity in root nodules is greater than the requirement of molybdenum for the activity of nitrate reductase in the vegetative tissues (18). Because nitrate is the major form of soil nitrogen absorbed by plant roots (19), the role of molybdenum as a functional component of nitrate reductase is of greater importance in plant nutrition than its role in N 2 fixation. Like other molybdenum enzymes in plants, nitrate reductase is a homodimeric protein. Each identical subunit can function independently in nitrate reduction (9), and each consists of three functional domains: the N-terminal domain associated with a molybdenum cofactor (Moco), the central heme domain (cytochrome b 557 ), and the C-terminal FAD domain (7,20). This enzyme occurs in the cytoplasm and catalyzes the reduction of nitrate to nitrite (NO 2 Ϫ ) in plants (19): NO 3 Ϫ ϩ 2H ϩ ϩ 2e 2 Ϫ → NO 2 Ϫ ϩ 2H 2 O Nitrate and molybdenum are both required for the induction of nitrate reductase in plants, and the enzyme is either absent (21), or its activity is reduced (22), if either nutrient is deficient. In deficient plants, the induction of nitrate reductase activity by nitrate is a slow process, whereas the induction of enzyme activity by molybdenum is much faster (10). It has been demonstrated that the molybdenum requirement of plants is higher if they are supplied nitrate rather than ammonium (NH 4 ϩ ) nutrition (23)—an effect that can be almost completely accounted for by the molybdenum in nitrate reductase (12). 13.1.2.3 Xanthine Dehydrogenase In addition to the enzymes nitrogenase and nitrate reductase, molybdenum is also a functional compo- nent of xanthine dehydrogenase, which is involved in ureide synthesis and purine catabolism in plants (8). This enzyme is a homodimeric protein of identical subunits, each of which contains one molecule of FAD, four Fe-S groups, and a molybdenum complex that cycles between its Mo(VI) and Mo(IV) oxidation states (9,13). Xanthine dehydrogenase catalyzes the catabolism of purines to uric acid (7): purines → xanthine → uric acid In some legumes, the transport of symbiotically fixed N 2 from root to shoot occurs in the form of ureides, allantoin, and allantoic acid, which are synthesized from uric acid (10). Although xanthine Molybdenum 377 CRC_DK2972_Ch013.qxd 6/6/2006 1:13 PM Page 377 dehydrogenase is apparently not essential for plants (10), it can play a key role in nitrogen metabo- lism for certain legumes for which ureides are the most prevalent nitrogen compounds formed in root nodules (9). The poor growth of molybdenum-deficient legumes can be attributed in part to poor upward transport of nitrogen because of disturbed xanthine catabolism (10). 13.1.2.4 Aldehyde Oxidase Aldehyde oxidases in animals have been well characterized, but only recently has this molybdoen- zyme been purified from plant tissue and described (24). In plants, aldehyde oxidase is considered to be located in the cytoplasm where it catalyzes the final step in the biosynthesis of the phytohor- mones indoleacetic acid (IAA) and abscisic acid (ABA) (8). These hormones control diverse processes and plant responses such as stomatal aperture, germination, seed development, apical dominance, and the regulation of phototropic and gravitropic behavior (25,26). Molybdenum may therefore play an important role in plant development and adaptation to environmental stresses through its effect on the activity of aldehyde oxidase, although other minor pathways exist for the formation of IAA and ABA in plants (7). 13.1.2.5 Sulfite Oxidase Molybdenum may play a role in sulfur metabolism in plants. In biological systems the oxidation of sulfite (SO 3 2Ϫ ) to sulfate (SO 4 2Ϫ ) is mediated by the molybdoenzyme, sulfite oxidase (10). Although this enzyme has been well studied in animals (27), the existence of sulfite oxidase in plants is not well established. Marschner (9) explains that the oxidation of sulfite can be brought about by other enzymes such as peroxidases and cytochrome oxidase, as well as a number of metals and superox- ide radicals. It is therefore not clear whether a specific sulfite oxidase is involved in the oxidation of sulfite in higher plants (28) and, consequently, also whether molybdenum is essential in higher plants for sulfite oxidation. 13.2 DIAGNOSIS OF MOLYBDENUM STATUS OF PLANTS 13.2.1 D EFICIENCY The discovery of molybdenum as a plant nutrient led to the diagnosis of the deficiency in a number of crop plants, with the first report of molybdenum deficiency in the field being made by Anderson (29) for subterranean clover (Trifolium subterraneum L.). The critical deficiency concentration in most crop plants is quite low, normally between 0.1 and 1.0 mg Mo kg Ϫ1 in the dry tissue (12). Symptoms of molybdenum deficiency are common among plants grown on acid mineral soils that have low concentrations of available molybdenum, but plants may occasionally become deficient in peat soils due to the retention of molybdenum on humic acids (19,30). Plants also may be prone to molybdenum deficiency under low temperatures and high nitrogen fertility (31). Because molybdenum is highly mobile in the xylem and the phloem (32), its deficiency symp- toms often appear on the entire plant. This appearance is unlike many of the other essential micronutrients where deficiency symptoms are manifest primarily in younger portions of the plant. Molybdenum deficiency is peculiar in that it often manifests itself as nitrogen deficiency, particu- larly in legumes. These symptoms are related to the function of molybdenum in nitrogen metabo- lism, such as its role in N 2 fixation and nitrate reduction. However, plants suffering from extreme deficiency often exhibit symptoms that are unique to molybdenum. Legumes often require more molybdenum than other plants, particularly if they are dependent on N 2 as a source of nitrogen (9). Molybdenum-deficient legumes commonly become chlorotic, have stunted growth, and have a restriction in the weight or quantity of root nodules (33,34). In dicotyledonous species, a drastic reduction in leaf size and irregularities in leaf blade formation (whip- tail) are the most typical visible symptoms, caused by local necrosis in the tissue and insufficient 378 Handbook of Plant Nutrition CRC_DK2972_Ch013.qxd 6/6/2006 1:13 PM Page 378 differentiation of vascular bundles at an early stage of leaf development (35). Marginal and interveinal leaf necrosis is a symptom of extreme molybdenum deficiency, and symptoms are often associated with high nitrate concentrations in the leaf, indicating that nitrate reductase activity is impaired (12). The whiptail disorder is observed often in molybdenum-deficient cauliflower (Brassica oleracea var. botrytis L.), one of the most sensitive cruciferous crops to low molybdenum nutrition (36). In addition, molybdenum-deficient beans (Phaseolus vulgaris L.) often develop scald, where the leaves are pale with interveinal and marginal chlorosis, followed by burning of the leaf margin (36,37). In molybdenum-deficient tomatoes, lower leaves appear mottled and eventually cup upward and develop marginal necrosis (3). Molybdenum deficiency also decreases tasseling and inhibits anthesis and pollen formation in corn (Zea mays L.) (38). The inhibition of pollen forma- tion with molybdenum deficiency may explain the lack of fruit formation in molybdenum-deficient watermelon (Citrullus vulgaris Schrad.) (9,39). 13.2.2 EXCESS Most plants are not particularly sensitive to excessive molybdenum in the nutrient medium, and the crit- ical toxicity concentration of molybdenum in plants varies widely. For instance, molybdenum is toxic to barley (Hordeum vulgare L.) if leaf tissue levels exceed 135 mg Mo kg Ϫ1 (40), but crops such as cauliflower and onion (Allium cepa L.) are able to accumulate upwards of 600 mg Mo kg Ϫ1 without exhibiting symptoms of toxicity (41). However, tissue concentrations Ͼ500mg Mo kg Ϫ1 can lead to a toxic response in many plants (42), which is characterized by malformation of the leaves, a golden-yel- low discoloration of the shoot tissues (9), and inhibition of root and shoot growth (43). These symp- toms may, in part, be the result of inhibition of iron metabolism by molybdenum in the plant (12). Toxicity symptoms in plants under field conditions are very rare, whereas toxicity to animals feeding on forages high in this element is well known (44). A narrow span exists between nutritional deficiency for plants and toxicity to ruminants (45). Molybdenum concentrations Ͼ10mg Mo kg Ϫ1 (dry mass) in forage crops can cause a nutritional disorder called molybdenosis in grazing rumi- nants (9). This disorder is a molybdenum-induced copper deficiency that occurs when the consumed molybdate (MoO 4 2Ϫ ) reacts in the rumen with sulfur to form thiomolybdate complexes, which inhibit copper metabolism (46). Agricultural practices that can be used to decrease ruminant susceptibility to molybdenosis include field applications of copper and sulfur. The strong depressive effects of SO 4 2Ϫ on MoO 4 2Ϫ uptake can lower the molybdenum concentration in plants to levels that are nontoxic (47). Increasing the copper content of forages through fertilization may also help to reduce molybdenum- induced copper deficiency in animals (46). 13.2.3 MOLYBDENUM CONCENTRATION AND DISTRIBUTION IN PLANTS The requirement of plants for molybdenum is lower than any other mineral nutrient except nickel (Ni) (9). Plants differ in their ability to absorb molybdenum from the root medium (48), and the sufficiency range for molybdenum in plants varies widely (Table 13.1). Most plants contain sufficient levels of molybdenum—in the range of 0.2 to 2.0 mg Mo kg Ϫ1 —in their dry tissue, but the difference between the critical deficiency and toxicity levels can vary up to a factor of 10 4 (e.g., 0.1 to 1000 mg Mo kg Ϫ1 dry mass) (9). The source of nitrogen supplied to plants influences their requirement for molybdenum. Nitrate- fed plants generally have a high requirement for molybdenum (66), but there are conflicting reports as to whether plants supplied with reduced nitrogen have a molybdenum requirement. Cauliflower developed symptoms of molybdenum deficiency when grown with ammonium salts, urea, glutamate, or nitrate, in the absence of molybdenum (20). However, Hewitt (67) suggested that the molybdenum requirement, in the presence of reduced nitrogen, may result from the effects of traces of nitrate derived from bacterial nitrification. When cauliflower plants were supplied ammonium sulfate and no Molybdenum 379 CRC_DK2972_Ch013.qxd 6/6/2006 1:13 PM Page 379 380 Handbook of Plant Nutrition TABLE 13.1 Deficient and Sufficient Concentrations of Molybdenum in Plants Mo Concentration (mg kg ϪϪ 1 dry mass) Crop or Plant Type Plant Part Sampled Deficient Sufficient Reference Agronomic Crops Alfalfa (Medicago sativa L.) Upper portion of tops; prior to Ͻ0.4 0.5–5.0 49, 50 blossom Barley (Hordeum vulgare L.) Whole tops; boot stage 0.09–0.18 51 Canola (Brassica napus L.) Mature leaves without petioles 0.25–0.60 52 Corn (Zea mays L.) Stems Ͻ0.12 1.4–7.0 53 Ear leaves; silk stage Ͻ1.1 54 Cotton (Gossypium hirsutum L.) Fully mature leaves; after bloom 0.6–2.0 55 Oats (Avena sativa L.) Whole tops 0.2–0.3 52 Peanuts (Arachis hypogaea L.) Upper fully developed leaves Ͻ1 0.5–1.0 55, 56 Red clover (Trifolium pratense L.) Total aboveground plants; bloom Ͻ0.15 0.3–1.59 50 Whole plants; bud stage 0.46–1.08 41, 57 Rice (Oryza sativa L.) Upper fully developed leaves; 0.4–1.0 55 prior to flowering Soybeans [Glycine max (L.) Merr.] Whole plants Ͻ0.2 58 Upper fully developed leaves; 0.5–1.0 55 end of blossom Sugar beet (Beta vulgaris L. Leaf blades Ͻ0.16 0.2–20.0 59 ssp. vulgaris) Fully developed leaf without stem Ͻ0.15 0.2–20.0 50, 59 Sunflower (Helianthus annuus L.) Mature leaves from new growth 0.25–0.75 52 Tobacco (Nicotiana tabacum L.) Mature leaves from new growth 0.1–0.6 52 Wheat (Triticum aestivum L.) Whole tops; boot stage 0.09–0.18 51 Vegetable Crops Beans (Phaseolus vulgaris L.) Youngest fully expanded leaf; Ͻ0.2 0.2–5.0 36 flowering Beets (Beta vulgaris L.) Tops; 8 weeks old Ͻ0.06 60 Young mature leaves 0.15–0.6 36 Broccoli (Brassica oleracea L. Tops; 8 weeks old Ͻ0.05 60 convar. botrytis) Mature leaves from new growth 0.30–0.50 52 Cabbage (Brassica oleracea L. Wrapper leaves Ͻ0.3 0.3–3.0 36, 52 var. capitata) Carrots (Daucus carota L.) Mature leaves from new growth 0.5–1.5 52 Cauliflower (Brassica oleracea Young leaves showing whiptail 0.07 58 convar. botrytis var. botrytis) Aboveground portion of plants; Ͻ0.26 0.68–1.49 61 appearance of curd Cucumber (Cucumis sativus L.) Youngest fully mature leaves Ͻ0.2 0.2–2.0 36 Lettuce (Lactuca sativa L.) Leaves Ͻ0.07 0.08–0.14 41, 62 Onion (Allium cepa L.) Whole tops; maturity Ͻ0.06 Ͼ0.1 63 Pea (Pisum sativum L.) Recent fully developed leaves; 0.4–1.0 55 onset of blossom Potato (Solanum tuberosum L.) Leaf blades Ͻ0.16 64 Fully developed leaves; early bloom 0.2–0.5 55 CRC_DK2972_Ch013.qxd 6/6/2006 1:13 PM Page 380 molybdenum under sterile conditions, Hewitt and Gundry (68) found that plants showed no abnor- malities and apparently had no molybdenum requirement. On transfer to nonsterile conditions, whip- tail symptoms appeared as a characteristic symptom of molybdenum deficiency. Hewitt (17) later stated that molybdenum is of very little importance for some plants if nitrate reduction is not neces- sary for nitrogen assimilation, but that it is impossible to say that an element is not required by plants given the limits of current analytical techniques. Molybdenum is absorbed by plant roots in the form of the molybdate ion (MoO 4 2Ϫ ), and its uptake is considered to be controlled metabolically (19). In long-distance transport in plants, molybdenum is readily mobile in the xylem and phloem (32). The form in which molybdenum is translocated is unknown, but its chemical properties indicate that it is most likely transported as MoO 4 2Ϫ rather than in a complexed form (9). The proportion of various molybdenum constituents in plants naturally depends on the quantity of molybdenum absorbed and accumulated in the tissue. Molybdenum-containing enzymes, such as nitrogenase and nitrate reductase, constitute a major pool for absorbed molybdenum, but under conditions of luxury consumption, excess molybdenum can also be stored in the vacuoles of peripheral cell layers of the plant (69). The allocation of molybdenum to the various plant organs varies considerably among plant species, but generally the concentration of molybdenum is highest in seeds (12) and in the nodules of N 2 -fixing plants (9). However, when molybdenum is limiting, preferential accumulation in root nodules may lead to considerably lower molybdenum content in the shoots and seeds of nodulated legumes (70). Molybdenum concentrations in leaves have been found to exceed concentrations in the stems of sev- eral crop species such as tomato, alfalfa (Medicago sativa L.), and soybeans (Glycine max Merr.) (12). Molybdenum 381 TABLE 13.1 ( Continued ) Mo Concentration (mg kg ϪϪ 1 dry mass) Crop or Plant Type Plant Part Sampled Deficient Sufficient Reference Fruit Crops Apple (Malus sylvestris Mill.) Mature leaves from new growth 0.10–2.00 52 Avocado (Persea americana Mill.) Mature leaves from new flush 0.05–1.0 52 Orange (Citrus sinensis L.) Mature leaves from nonfruiting 0.1–0.9 52 Pear (Pyrus communis L.) Mid-shoot leaves from new growth 0.10–2.0 52 Peach (Prunus persica L. Batsch.) Mid-shoot leaves 1.6–2.8 52 Strawberry (Fragaria x Mature leaves from new growth 0.25–0.50 52 ananassa Duch.) Ornamental Plants New Guinea impatiens Mature leaves from new growth 0.15–1.0 52 (Impatiens x hybrids) Poinsettia (Euphorbia Mature leaves from new growth Ͻ0.5 0.12–0.5 52, 65 pulcherrima Willd.) Rose, hybrid tea (Rosa x Upper leaflets from mature leaves 0.1–0.9 52 cultivars) Salvia (Salvia splendens) Mature leaves from new growth 0.2–1.08 52 Snapdragon (Antirrhinum majus L.) Mature leaves from new growth 0.12–2.0 52 Verbena (Verbena x hybrids) Mature leaves from new growth 0.14–0.8 52 Trees and Shrubs Common lilac (Syringa vulgaris L.) Mature leaves from new growth 0.12–4.0 52 Douglass fir (Pseudotsuga menziesii) Terminal cuttings 0.02–0.25 52 Loblolly pine (Pinus taeda L.) Needles from terminal cuttings 0.12–0.56 52 Source: Adapted from U.C. Gupta, in Molybdenum in Agriculture, Cambridge University Press, New York, 1997, pp. 150–159. With permission from Cambridge University Press. CRC_DK2972_Ch013.qxd 6/6/2006 1:13 PM Page 381 13.2.4 ANALYTICAL TECHNIQUES FOR THE DETERMINATION OF MOLYBDENUM IN PLANTS The molybdenum status of crops is often overlooked by the farming community, probably because of the relatively low crop requirement for molybdenum and because of a lack of education on the necessity of molybdenum in fertility programs. In addition, many commercial soil and plant analy- sis laboratories fail to report this nutrient in routine tissue and soil analyses. This omission may be partially due to the difficulties in accurately determining the small quantities of molybdenum that are normally present in plant tissues. It is possible that many molybdenum deficiencies in crop plants are misdiagnosed as nitrogen deficiency because of the similarity in their deficiency symptoms. The two most common methods of molybdenum extraction from plant tissues are dry ashing (71) and wet digestion (72), both of which give similar results (12). Dry ashing is often the preferred method of extraction due to the potential hazards involved with the use of perchloric acid (HClO 4 ) for wet digestion (72). Several analytical techniques have been proposed for the determination of molybdenum in the resulting extracts including the dithiol and thiocyanate colorimetric methods, determination by atomic absorption spectrometry (AAS), graphite furnace atomic absorption spec- trometry (GF-AAS), and by inductively coupled plasma atomic emission spectrometry (ICP-AES). As the detection of molybdenum by ICP-AES is less sensitive than for other elements, this method should be used only for plant tissues suspected of having molybdenum concentrations Ͼ1.0mg Mo kg Ϫ1 (dry mass) (73,74). The dithiol colorimetric method and the AAS method are probably the most commonly used techniques for determining molybdenum in soil and plant materials (12). The dithiol method developed by Piper and Beckworth (75) and modified by Gupta and MacKay (76) is more sensitive and precise than other colorimetric methods used for the determi- nation of molybdenum in plant tissues. This method is based on precipitation and extraction of a green-colored molybdenum dithiol complex after removal of interfering ions from the test solution (77). The molybdenum concentration is determined by comparing the absorbance of the sample with known standards on a light spectrophotometer. The detection limit of the dithiol method is about 20 ng Mo mL Ϫ1 , and the recovery of molybdenum added to the plant material has been greater than 90% (12). Although this method is relatively inexpensive, the procedure may be too tedious and time-consuming for use in many commercial analytical laboratories. For procedures of the dithiol method, readers are referred to Gupta (73). Trace quantities of molybdenum in plant material have been determined by flame (78) or flameless AAS (79). These procedures provide adequate sensitivity for molybdenum and are rela- tively rapid, but are subject to matrix interferences (77). The GF-AAS method (80) improves the accuracy and precision of determining low concentrations of molybdenum, and the procedure is applicable to a range of different plant matrices (73). The detection limits for the determination of molybdenum by AAS using flame and graphite furnace are reported to be 10 and 2 ng mL Ϫ1 , respec- tively (78), and the recovery of molybdenum by these two methods is similar to that of the dithiol colorimetric method, ranging from 92 to 95% (12). For details of the flame and graphite furnace AAS methods, the reader is referred to Khan et al. (78) and Gupta (73). 13.3 ASSESSMENT OF MOLYBDENUM STATUS OF SOILS 13.3.1 S OIL MOLYBDENUM CONTENT The amount of naturally occurring molybdenum in soils depends on the molybdenum concentrations in the parent materials. Igneous rock makes up some 95% of the Earth crust (81) and contains ∼2mg Mo kg Ϫ1 . Similar amounts of molybdenum are present in sedimentary rock (82). The total molybde- num content of soils differs by soil type and sometimes by geographical region (Table 13.2). Soils nor- mally contain between 0.013 and 17.0 mg kg Ϫ1 total molybdenum (44), but molybdenum concentrations can exceed 300mg Mo kg Ϫ1 in soils derived from organic-rich shale (83). Large quan- tities of molybdenum also occur in soils receiving applications of municipal sewage sludge (84) or in soils that are polluted by mining activities (46). Most agricultural soils contain a relatively low amount 382 Handbook of Plant Nutrition CRC_DK2972_Ch013.qxd 6/6/2006 1:13 PM Page 382 Molybdenum 383 TABLE 13.2 Molybdenum Content of Surface Soils of Different Countries Soil Country Range (mg kg ϪϪ 1 dry weight) Podzols and sandy soils Australia 2.6–3.7 Canada 0.40–2.46 New Zealand 1–2 a Poland 0.2–3.0 Yugoslavia 0.17–0.51 b Russia 0.3–2.9 Loess and silty soils New Zealand 2.2–3.1 a China 0.4–1.1 Poland 0.6–3.0 United States 0.75–6.40 Russia 1.8–3.3 Loamy and clayey soils Great Britain 0.7–4.5 Canada 0.93–4.74 Mali Republic 0.5–0.75 New Zealand 2.1–4.2 a Poland 0.1–6.0 United States 1.2–7.2 United States c 1.5–17.8 Russia 0.6–4.0 Fluvisols India 0.4–3.1 b Czech Republic 2.8–3.5 Mali Republic 0.44–0.65 Yugoslavia 0.35–0.53 b Russia 1.8–3.0 Gleysols Australia 2.5–3.5 India 1.1–1.8 b Ivory Coast 0.18–0.60 Yugoslavia 0.52–0.74 Russia 0.6–2.0 Histosols and other organic soils Canada 0.69–3.2 Russia 0.3–1.9 Forest soils Bulgaria 0.3–4.6 Former Soviet Union 0.2–8.3 Various soils Great Britain 1–5 India 0.013–2.5 Italy 0.4–2.2 Japan 0.2–11.3 United States 0.8–3.3 Russia 0.8–3.6 a Soils derived from basalts and andesites. b Data for whole soil profiles. c Soils from areas of the western states of Mo toxicity to grazing animals. Source: From A. Kabata-Pendias, H. Pendias, Trace Elements in Soils and Plants. 3rd ed., CRC Press, Boca Raton, FL. 2001, pp. 260–267. Copyright CRC Press. CRC_DK2972_Ch013.qxd 6/6/2006 1:13 PM Page 383 of molybdenum by comparison, with an average of 2.0 mg kg Ϫ1 total molybdenum and 0.2 mg kg Ϫ1 available molybdenum (19). Soils derived from granite, organic-rich shale, or limestone, and those high in organic matter are usually rich in molybdenum (85,86), and the available molybdenum content generally increases with alkalinity or fineness of the soil texture (85). In contrast, molybdenum is often deficient in well- drained coarse-textured soils or in soils that are highly weathered or acidic (83,87). The accumulation of molybdenum varies with depth in the soil, but molybdenum is normally highest in the A horizons of well-drained soils and is highest in the subsoil of poorly drained mineral soils (83). In soils, molyb- denum can occur in four fractions: (a) dissolved molybdenum in the soil solution, (b) molybdenum occluded with oxides, (c) molybdenum as a mineral constituent, and (d) molybdenum associated with organic matter (85). 13.3.2 FORMS OF MOLYBDENUM IN SOILS The speciation and availability of molybdenum in the soil solution is a function of pH. At water pH Ͼ5.0, molybdenum exists primarily as MoO 4 2Ϫ (84), but at lower pH levels the HMoO 4 Ϫ and H 2 MoO 4 0 forms dominate (44). For each unit increase in soil pH above pH 5.0, the soluble molyb- denum concentration increases 100-fold (88). Plants preferentially absorb MoO 4 2Ϫ and therefore the molybdenum nutrition of plants can be manipulated by altering soil acidity. Soil liming is commonly used to alleviate molybdenum deficiencies in plants by increasing the quantity of plant-available molybdenum in the soil solution (89), but the effect of liming on molybdenum nutrition varies by soil and plant type (Table 13.3). Excessive lime use may decrease the solubility of molybdenum through the formation of CaMoO 4 (44), but Lindsay (90) suggests that this complex is too soluble to persist in soils. Using lime to change the acidity of a clay loam from pH 5 to 6.5 resulted in greater molyb- denum accumulation in cauliflower, alfalfa (Medicago sativa L.), and bromegrass (Bromus inermis Leyss.), but molybdenum accumulation was relatively unaffected if plants were grown in a sandy loam (Table 13.3) (87). For plants grown in sandy loam, lime and molybdenum were both required to significantly increase the molybdenum content of the plant tissue. 384 Handbook of Plant Nutrition TABLE 13.3 Effects of Soil pH on Molybdenum Concentration in a Few Crops Grown on Two Soils Mo concentration (mg kg ϪϪ 1 ) Cauliflower Alfalfa Bromegrass Soil pH a No Mo Mo (2.5 mg kg ϪϪ 1 ) No Mo Mo (2.5 mg kg ϪϪ 1 ) No Mo Mo (2.5mg kg ϪϪ 1 ) Silty clay loam 5.0 Trace 0.02 Trace 0.43 0.11 0.95 5.5 Trace 0.21 0.51 4.40 0.30 1.80 6.0 0.11 1.62 0.91 4.63 0.27 1.67 6.5 0.56 6.43 1.48 4.93 0.62 2.30 Culloden sandy loam 5.0 Trace 0.39 Trace 0.11 0.02 0.35 5.5 Trace 1.34 Trace 2.04 0.02 1.09 6.0 Trace 3.15 Trace 2.01 0.04 3.59 6.5 Trace 3.58 Trace 3.32 0.05 3.77 a Soil:water ratio 1:2. Source: From U.C. Gupta, in Molybdenum in Agriculture, Cambridge University Press, New York, 1997, pp. 71–91. Reprinted with permission from Cambridge University Press. CRC_DK2972_Ch013.qxd 6/6/2006 1:13 PM Page 384 [...]... CRC_DK2972_Ch 013. qxd 6/6/2006 1 :13 PM Page 388 388 Handbook of Plant Nutrition 13. 4.1.2 Foliar Fertilization Sodium molybdate and ammonium molybdate are the most commonly used molybdenum sources for foliar fertilization because of their high solubility in water Foliar applications of molybdenum are most effective if applied at early stages of plant development, and generally a 0.025 to 0.1% solution of sodium... J Lipsett Molybdenum in soils, plants, and animals Adv Agron 34:73–115, 1981 CRC_DK2972_Ch 013. qxd 390 6/6/2006 1 :13 PM Page 390 Handbook of Plant Nutrition 13 D Voet, J.G Voet Biochemistry 2nd ed New York: Wiley, Inc., 1995, pp 820–822 14 R.M Allen, J.T Roll, P Rangaraj, V.K Shah, G.P Roberts, P.W Ludden Incorporation of molybdenum into the iron-molybdenum cofactor of nitrogenase J Biol Chem 274:15869–15874,... 91–98 65 D.A Cox Foliar-applied molybdenum for preventing or correcting molybdenum deficiency of poinsettia Hortscience 8:894–895, 1992 CRC_DK2972_Ch 013. qxd 392 6/6/2006 1 :13 PM Page 392 Handbook of Plant Nutrition 66 S.C Agarwala, E.J Hewitt Molybdenum as a plant nutrient III The interrelationships of molybdenum and nitrate supply in the growth and molybdenum content of cauliflower plants grown in sand... content of pasture plants II Effect of soluble phosphates, available nitrogen and soluble sulfates Soil Sci 71:387–398, 1951 99 P.R Stout, W.R Meagher Studies of the molybdenum nutrition of plants with radioactive molybdenum Science 108:471–473, 1948 100 P.R Stout, W.R Meagher, G.A Pearson, C.M Johnson Molybdenum nutrition of crop plants I Influence of phosphate and sulfate on the absorption of molybdenum... absorption of radioactive molybdenum by tomatoes, and decreased molybdenum absorption by tomatoes (Lycopersicon esculentum Mill.) and peas (Pisum sativum L.) in soil (100) CRC_DK2972_Ch 013. qxd 6/6/2006 1 :13 PM Page 386 386 Handbook of Plant Nutrition 13. 3.4 SOIL ANALYSIS The use of soil testing to predict the soil’s capacity to supply molybdenum for plant growth can be difficult because of the relatively... (GFAAS) Commun Soil Sci Plant Anal 16:1279–1291, 1985 108 J.L Grigg The distribution of molybdenum in the soils of New Zealand I Soils of the North Island N.Z J Agric Res 3:69–86, 1960 109 I Barshad Factors affecting the molybdenum content of pasture plants I Nature of soil molybdenum, growth of plants, and soil pH Soil Sci 71:297– 313, 1951 110 U.C Gupta, D.C MacKay Extraction of water soluble copper... Foliar application of molybdenum in common bean II Nitrogenase and nitrate reductase activities in a soil of low fertility J Plant Nutr 21:2141–2151, 1998 118 R.F Vieira, E.J.B.N Cardoso, C Vieira, S.T.A Cassini Foliar application of molybdenum in common bean III Effect on nodulation J Plant Nutr 21:2153–2161, 1998 CRC_DK2972_Ch 013. qxd 394 6/6/2006 1 :13 PM Page 394 Handbook of Plant Nutrition 119 E.J... species A role for anthocyanins? Plant Physiol 126 :139 1–1402, 2001 70 J Ishizuka Characterization of molybdenum absorption and translocation in soybean plants Soil Sci Plant Nutr 28:63–78, 1982 71 R.O Miller High-temperature oxidation: dry ashing In: Y.P Kalra, ed Handbook of Reference Methods for Plant Analysis Boca Raton, FL: CRC Press, 1998, pp 53–56 72 R.O Miller Nitric-perchloric acid wet digestion... accumulation of molybdenum in organic matter can be particularly high if soil drainage is impeded (95) Organic-matter-rich soils can supply adequate amounts of molybdenum for plant growth due to a slow release of molybdenum from the organic complex (44) However, there are conflicting reports concerning the effect of soil organic matter on the availability of molybdenum in the soil solution Plant- available... testing and Plant Analysis, 2nd ed Madison, WI: SSSA, 1973, pp 271–278 60 C.M Johnson, G.A Pearson, P.R Stout Molybdenum nutrition of crop plants II Plant and soil factors concerned with molybdenum deficiencies in crop plants Plant Soil 4:178–196, 1952 61 E.W Chipman, D.C MacKay, U.C Gupta, H.B Cannon Response of cauliflower cultivars to molybdenum deficiency Can J Plant Sci 50:163–167, 1970 62 W Plant The . Nitrogenase 376 13. 1.2.2 Nitrate Reductase 377 13. 1.2.3 Xanthine Dehydrogenase 377 13. 1.2.4 Aldehyde Oxidase 378 13. 1.2.5 Sulfite Oxidase 378 13. 2 Diagnosis of Molybdenum Status of Plants 378 13. 2.1 Deficiency. essential in higher plants for sulfite oxidation. 13. 2 DIAGNOSIS OF MOLYBDENUM STATUS OF PLANTS 13. 2.1 D EFICIENCY The discovery of molybdenum as a plant nutrient led to the diagnosis of the deficiency. plants were supplied ammonium sulfate and no Molybdenum 379 CRC_DK2972_Ch 013. qxd 6/6/2006 1 :13 PM Page 379 380 Handbook of Plant Nutrition TABLE 13. 1 Deficient and Sufficient Concentrations of