CASE STUDY 16.1  Boron toxicity and Australian agriculture

Jeff Paull

Boron (B) is an essential micronutrient, but is potentially toxic to plants over much of southern Australia where high concen-trations of soil B reduce crop yields, especially in sensitive varieties.

Toxic levels of soil B can derive from over-application of B when correcting a deficiency, contamination in industrial and sewerage sludges, high concentrations in groundwater used for irrigation and high levels in parent material, including marine sediments. Seawater normally contains appreciable levels of B and as a consequence deposits that remain after ancient seas recede contain high levels of B and other soluble salts. As these deposits weather, B is released and slowly redistributed by leaching.

Boric acid, B(OH)₃, is the main form of free B in soil, and of much lower solubility than the accompanying sodium chloride in soils derived from these marine sediments. As a consequence, B has not been leached from root zones in low- rainfall environments of southern Australia. In addition, hydroxyl groups of boric acid react with clay particles and remain bound under alkaline conditions, so that leaching is further decreased. Geographic regions with high soil B usually correspond to areas of marine sediments, rich in clay and subject to low rainfall.

Soil B in southern Australia has been determined from analysis of soil cores, plant shoots, harvested grain and from observation of B toxicity. Visible symptoms on leaf tips of barley include distinctive black spots within dead leaf tissue. High-B soils occur on Eyre Peninsula, mallee regions of South Australia and Victoria and low-rainfall regions of inland southeastern Western Australia. B toxicity is very widespread, and levels are generally highest in subsoils rather than root zones. Soil amelioration is thus an impractical solution, so that most research efforts have been directed towards selection of B-tolerant crop varieties.

Genetic variation in crop response to B has been identified (Figure 1). Similar variation has also been observed for several other major crops of southern Australia, including barley, oats and peas. While there is some variation in B tolerance among Australian varieties of these crops, more tolerant lines have been identified in collections from the Middle East, India, Japan and parts of South America. In all cases, these B-tolerant crops have adapted to soils subject to either marine or volcanic influence.

Tolerance to high concentrations of B is under the control of a number of major genes, and segregation ratios observed in F₂ and F₃ generations conform to the Mendelian ratio of 1/4 homozygous sensitive to 1/2 heterozygous to 1/4 homozygous tolerant. Two examples of segregation ratios in wheat shown here compare observed ratios (Obs) with expected ratios (Exp); Chi-square analysis (χ²) confirms that observed frequencies agree with Mendelian prediction:

		Sensitive	Heterozygous	Tolerant	χ2
Warigal × (Wl*MMC)	Obs	27	63	32
	Exp	30.5	61	30.5	0.34
Halberd × Warigal	Obs	27	63	20
	Exp	27.5	55	27.5	2.80

These genes act in an additive manner and tolerant varieties have dominant alleles at more loci than do sensitive varieties.

Plant response to B is thus subject to control by major genes, so that genes for tolerance can be transferred to sensitive varieties by backcrossing. This involves hybridisation between a sensitive, but otherwise well-adapted, variety and a tolerant variety. Progeny are tested for response to B and tolerant selections are then crossed to the sensitive variety. Progeny of this cross are again tested for response to B and tolerant plants selected. It is possible to reconstitute the agronomic traits of the sensitive variety, but now with tolerance to B, by repeating this cycle a number of times. In one such outcome (Figure 1) three cycles of backcrossing transferred B tolerance from Halberd to the sensitive variety Schomburgk. Halberd was the most widely grown variety in Australia during the 1970s and 1980s, but more recent varieties, such as Schomburgk, have higher yield potential and better flour quality. Both tolerant and sensitive plants occurred among the progeny and a tolerant selection resulted in the new variety BT-Schomburgk.

Mechanisms responsible for root acquisition of B are not known, but B uptake is known to be closely correlated with water use and is higher under conditions conducive to rapid transpiration. Once absorbed, B moves with the transpiration stream and concentrates at xylem endings. Concentration gradients thus develop within leaves, and overall leaf concen-tration will vary with transpirational history. B concentrations are generally highest at leaf tips or along margins, depending upon venation, and are higher in old leaves compared with young leaves. For example, B concentration in the terminal
4 cm of a barley leaf, when grown under high B conditions, was 2493 mg kg^–1 (dry mass), while the corresponding concen-tration for a segment 14–20 cm from the tip was only 82 mg kg^–1. Average concentration for the whole leaf was 208 mg kg^–1 (J.G. Paull, unpublished data).

Visible symptoms of B toxicity coincide with regions of highest B concentration and consist initially of chlorosis followed by tissue death. Varieties that tolerate high concen-trations of B (Figure 1) are able to exclude B from their roots. They therefore have lower shoot concentrations (Figure 2) and exhibit less severe symptoms of B toxicity compared with more sensitive varieties.

In retrospect, B tolerance was probably a major factor influencing progeny selection from breeding programs for wheat varieties in Australia. Several varieties have dominated Australian wheat production in southern Australia during the twentieth century and the majority of these are tolerant to B. On the other hand, varieties grown in New South Wales and Queensland, where high concentrations of B do not occur, are invariably sensitive to soil B. For example, the variety Halberd, which is one of the most tolerant Australian varieties, was the most widely grown variety in southern Australia during the 1970s and 1980s. Cultivation of Halberd was most concentrated in areas of high soil B. At some silos on Eyre Peninsula, Victorian and South Australian mallee regions as well as inland Western Australia, more than 70% of the total grain receivals were Halberd. By contrast, in low-B regions, Halberd accounted for less than 20% of production. By implication, any successful field crops in southern Australia will have some measure of B tolerance.

References

Nable, R.O. (1988). ‘Resistance to boron toxicity amongst several barley and wheat cultivars: a preliminary examination of the resistance mechanism’, Plant and Soil, 112, 45–52.

Paull, J.G., Rathjen, A.J. and Cartwright, B. (1991). ‘Major gene control of tolerance of bread wheat (Triticum aestivum L.) to high concentrations of soil boron’, Euphytica, 55, 217–228.

 

16.3.1  Essential mineral nutrients

Solution culture experiments have established a number of facts basic to plant growth on soils — that plant roots must have a supply of oxygen, that solid soil particles and micro-organisms, while sometimes beneficial, are not essential, that in addition to C, H and O some 14 or 15 chemical elements are essential for plant growth, and that all of these essential elements may be supplied to plant roots as simple ions of inorganic salts in solution and must be supplied in adequate but non-toxic amounts.

A chemical element is regarded as essential if, in its absence, a plant cannot complete its life cycle. An alternative criterion has also been suggested — that the element is part of an essential plant constituent or molecule. So far, only the first criterion has been used to establish essentiality although the latter has proved useful as a guide to further research, as, for example, in the association of Mn with laccase activity and Ni with urease activity.

‘Essential mineral nutrients’ or simply ‘essential nutrients’ include all those chemical elements which are normally absorbed from the soil solution by higher plants. They exclude C, H, and O which comprise more than 99% of metabolically active leaves and some 90–95% of their dry matter, but, paradoxically, they include N which does not occur in any soil mineral and is supplied to some plants from air. Essential mineral nutrients comprise less than 1% of fresh mass in active leaves and 5–10% of their dry matter; yet within this small proportion, all 14 or 15 elements must be present in adequate amounts for healthy growth and effective reproduction.

Functions of nutrients

Historically, essential nutrients have been classified in one of two groups based on amounts required by plants, namely macronutrients or micronutrients, and that convention has been adopted for convenience in Table 16.2. This distinction reflects historical sequence and experimental difficulties in discovering essential nutrients. Following advances in nine-teenth century chemistry, essentiality of macronutrients was relatively easy to prove, but essentiality of micronutrients (except Fe) was elusive, requiring great care in eliminating contamination from macronutrient salts, from water and from other environmental sources (see Feature essay 16.1). The classification remains in common use, and was therefore used in Table 16.2 but the distinction between macro and micro is somewhat arbitrary. In terms of chemistry and function, an alternative grouping is outlined below.

Group 1: N, S

N and S are covalently bound in organic compounds in reduced states. They are essential components of proteins providing reactive groups for interaction with metabolites and other nutrients. They are generally absorbed as oxyanions which, except for small amounts of sulphate in a few organic com-pounds, must be reduced before use.

Group 2: P, B

P and B are covalently bound in organic compounds in their fully oxidised states. P is present in phospholipids of cell membranes, nucleic acids of chromosomes and a large number of intermediary metabolites including many in which it plays a crucial role in bioenergetics. P is always present as mono- or polyorthophosphate which behaves as a weak acid. B, like P, is thought to function only in its fully oxidised form; B oxyacid is much weaker and is largely undissociated at the pH of cells. B complexes strongly with hydroxyls in adjacent cis-diol configuration in sugars and their derivatives such as those in the hemicellulose of cell walls, but key metabolic or structural functions still remain unknown.

Group 3: K, Ca, Mg, Na, Cl

K, Ca, Mg, Na and Cl are present primarily in ionic form as either free ions in solution or as entities reversibly adsorbed by electrostatic forces on charged sites. K, Ca and Mg are required in relatively large amounts while Na and Cl, though often present in large amounts, are required only in trace amounts.

Group 3 nutrients function in osmotic adjustment of cell activities and in enzyme activation, probably by modifying the shape and orientation of substrates and enzymes. Ca and to a lesser extent Mg also stabilise cell structures such as membranes and cell walls, probably via their divalent positive charge which forms cross-links with negative charges on cell structures. Some Ca binds strongly and reversibly to the protein calmodulin which in turn activates several enzymes and controls Ca transport. Some Mg is structurally bound in chlorophyll (a strict 1:1 stoichiometry of Mg atoms to chlorophyll molecules is universal)

Group 4: Mn, Fe, Co, Ni, Cu, Zn, Mo

Fe, Co, Ni, Cu, Zn, Mo and, to some extent, Mn are tightly bound in proteins as metalloproteins or, in the case of Co, which is only required in plants fixing N₂, in a coenzyme. All are transition series elements, and with the exception of Zn all show variation in valency state with attendant variation in physiological effect. Group 4 elements govern a wide range of reactions including many oxidation–reductions in which all except Zn can act as electron carriers by undergoing reversible oxidation–reduction. All are micronutrients, and all except Mn form strong complexes with organic and amino acids. Mn is exceptional in that it resembles Group 3 nutrients in being present largely as a free divalent ion which can replace Mg
in the activation of a number of enzymes. But some Mn is tightly bound within the water-splitting apparatus of photo-system II and in superoxide dismutase (Section 1.2.3), an enzyme system that helps dissipate harmful effects of singlet oxygen formation. Mn deficiency results in highly charac-teristic induction kinetics for in vivo chlorophyll a fluorescence (Figure 16.3) and can be used as a diagnostic tool in combination with chemical assay.

16.3.2  Quantitative requirements

For practical use in crop production, plant nutritionists need to know whether a plant’s internal supply of a nutrient is optimal for growth and, if not, how best to make it so. Answers to these two questions characterise a plant’s nutrient requirements.

Unfortunately, discussions about a plant’s quantitative nutrient requirements frequently fail to differentiate between the two quite distinct entities of internal and external requirements. Such confusion can be avoided with appropriate terminology: ‘internal requirement’ is the minimal concentration of a nutrient required within plant tissues and organs to sustain optimal metabolism and growth; ‘external requirement’ is the amount of a nutrient which must be supplied to soil or other culture medium for a plant to meet the internal requirement.

Estimates of internal nutrient requirements of actively metabolising leaves of dicotyledonous plants (Table 16.2) are minimal concentrations for maximal growth. Internal require-ments of roots for Mn and Mg could be expected to be lower than those for leaves, while those of N₂-fixing nodules for Co, which is not required by leaves, is around 0.1 mg kg^–1.

Internal nutrient requirements of grasses and other monocotyledons appear similar to those of dicotyledons, except for a halving of their Ca and B requirements. With these exceptions and differences in the extent to which plants can use Na to replace K, variation in internal nutrient requirements offers little scope for improving nutrient use efficiency of metabolic processes by selection or genetic mani-pulation. By contrast, wide variation in shoot tissue concentrations of micronutrient ions acquired by different genotypes does occur (Figure 16.4) and most notably for Mn (Figure 16.4). Such gene-based differences in acquisition of soil nutrients does constitute a ready source of variation in uptake efficiency that can be put to good effect in breeding cultivars better adapted to deficient soils (see also the frontispiece to Chapter 16).

External requirements of seedlings may be modified by seed nutrient content, and in some cases seeds may contain sufficient Mo and Co for the life cycle of annual plants.

Soil nutrient resources will vary in their availability to plants due to physico-chemical conditions in root zones, as well as fluctuations in root activity. Such intermittency in supply is buffered within plants by redistribution of internal nutrient resources as excess amounts in older tissues are retranslocated to growing regions. Significantly, not all nutrients can be recycled in this way, and in many plants Ca, B and sometimes Fe and Mn must be supplied continuously. Very little Ca or B moves from leaves to growing regions. Indeed, roots cannot grow into environments lacking Ca or B, even when other parts of the same root system have ample supplies of these nutrients. Internal remobilisation of Fe and Mn embedded within plant tissues is similarly restricted.

This failure of Ca, B, Fe and Mn to move from leaves to growing regions has been attributed to their low phloem mobility. While this is probably true for Ca, restricted movement of B, Fe and Mn is better attributed more broadly to their immobility after deposition in leaves. Immobile nutrients such as Ca contrast sharply with highly mobile nutrients such as N, P and K. Several other nutrients including S, Cu and Zn display variable mobility from leaves, being immobile from actively metabolising leaves but moving out of senescing leaves (Table 16.2). B physiology remains an enigma with some species showing B mobility in association with sorbitol translocation. Those species translocating sucrose typically show low B mobility.

CASE STUDY 16.2  Nutrient response in Eucalyptus grandis

Robin Cromer

Eucalyptus grandis (flooded gum or rose gum) is native to high-rainfall and nutrient-rich sites in warm–temperate and sub-tropical zones along the east coast of Australia. This species has evolved in situations where fast growth conferred a selective advantage and that adaptative feature of this species has been used in commercial plantation forests around the world.

Data reported here represent part of a broader project to examine the feasibility of growing commercial plantations of E. grandis in Queensland for production of pulpwood. Experiments were established to determine the maximum production of stemwood that could be achieved where limitations due to nutrients and water were eliminated. Maintenance of a highly favourable environment for tree growth also provides ideal conditions for prolific growth of competing weeds, so early canopy closure to facilitate ‘capture’ of the site by the tree crop and thus suppress weeds was a major objective.

A range of nutrient treatments was applied at regular intervals during the first three years after planting to determine optimum rates for tree growth. In this case study, however, only results showing the highest rate of fertiliser application, with and without supplemental irrigation, are highlighted. In addition to a basal dressing containing K, S, Cu, Zn and B, a total of 1540 kg ha^–1 of N with 460 kg ha^–1 of P was applied in the heaviest treatment. This treatment was used to ensure nutrients did not limit growth, recognising that such high rates were far beyond economic levels of nutrient application. Irrigation was applied so that total natural precipitation plus irrigation was approximately equal to Class A pan evaporation.

Figure 1(a), (b) shows a stand of E. grandis aged 4.5 years at Toolara, near Gympie in Queensland, which received regular applications of N and P fertiliser (+F) during the first three years after the trees were planted. When the photograph was taken, the standing volume of stemwood was 160 m³ ha^–1, compared with only 44 m³ ha^–1 (see Figure 1b) for a nearby stand of the same age which received no fertiliser (–F).

Leaf area index (LAI) in +F stands increased rapidly to 4.5 during the first year after planting, and then became relatively stable at about 5 (Figure 2a). By comparison, LAI in –F stands increased quite slowly for about 18 months and then stabilised around 1.

These marked differences in LAI were nutrient driven and had a major impact on accumulation of stemwood volume during the first three years of tree growth (Figure 2b; Cromer et al. 1993). Trees did not experience water stress during the first 2.5 years after planting so there was no response to irrigation and data for irrigation treatments have not been presented. A dry period after that time resulted in some reduction of LAI in +F plots (to about 4, Figure 2a) but there was no significant response to irrigation until three or more years of age.

Substantial differences in stemwood volume between +F and –F stands developed over the first three years after planting. However, large early differences in LAI were a major determinant of subsequent growth, and were clearly established well before the first measurement at 0.66 years (Figure 2a). Studies with seedlings and saplings (Section 6.2) indicate that changes in specific leaf area (SLA) due to nutrition are accompanied by greater photosynthetic capacity. Both features are important determinants of growth rate in seedlings and small trees but their influence declines rapidly as trees approach canopy closure.

Related observations on canopy extent and plantation biomass showed a strong relationship between net primary production (NPP) and mean LAI in each year of this three-year study. The relationship between NPP and LAI was independent of nutrient treatment, and demonstrated that area of foliage available to intercept radiant energy rather than canopy nutrient status was the more important factor driving productivity (see Section 12.4.2 for further discussion on underlying principles). In effect, application of fertiliser increased LAI substantially thus enabling useful interception of a high proportion of incident sunlight. After canopy closure, high radiation levels in this subtropical environment enabled +F stands to intercept in excess of 4 GJ m^–2 year^–1 of photosynthetically active radiation, driving NPP at rates of about 30 t ha^–1 year^–1. Such levels of wood production rank among the highest values ever reported worldwide (Miller 1989) and are particularly impressive for such a young plantation (trees less than three years old).

Positive interactions between genotype and nutrient supply have been observed in plantation foresty where trees from families characterised by inherently faster growth have showed a stronger response to added nutrients. To ensure that full expression of growth responses would be recorded in these present trials, seedlings were sourced from four different seedlots of E. grandis. A significant interaction between seedlot and fertiliser treatment was observed. Seedlots selected from plan-tations within the general climatic zone of the experiments (Coffs Harbour and Pomona) generally performed well and responded more strongly to fertiliser, whereas seed collected from native forest stands further south (Bulahdelah) or further north (Atherton) performed relatively poorly and did not respond so well to higher nutrient levels. By implication, superior genetic material could be selected for superior per-formance under well-nourished plantation conditions. As an extra bonus, the Pomona seedlot also responded to irrigation in combination with fertiliser after three years of age, such that this combination produced outstanding growth with a mean annual increment in stemwood volume of 50 m³ ha^–1 year^–1 once trees were five years old (Cromer 1995).

References

Cromer, R.N. (1995). ‘Fast-growing sub-tropical eucalypt plantations’, Onwood, 11 (Summer 1995–96), 4–5.

Cromer, R.N., Cameron, D.M., Rance, S.J., Ryan, P. and Brown, M. (1993). ‘Response to nutrients in Eucalyptus grandis: I Biomass accumulation’, Forest Ecology and Management,
62, 211–230.

Miller, H.G. (1989). ‘Internal and external cycling of nutrients in forest stands’, in Biomass Production by Fast-Growing Trees, eds J.S. Pereira and J.J. Landsberg, 73–80, Kluwer Academic Publications: Dordrecht.

 

 

 

16.3.3  Deficiencies and responses

The early German soil chemist Liebig postulated that plants growing on a nutrient-deficient soil responded in direct proportion to the addition of the deficient nutrient. A later German worker, Mitscherlich, showed that responses more frequently followed the economists’ Law of Diminishing Returns with successive additions of a deficient nutrient giving progressively smaller increments of growth approaching a plateau of maximum yield. His ‘Mitscherlich Equation’ for representing response curves provides a good fit to the ascending asymptote of many experimental data, but does not describe a supraoptimal depression in plant growth which follows the yield plateau with excess nutrient supply (Figure 16.5).

Early German workers (Sprengel and later Liebig) recognised that plants require an adequate supply of all essential nutrients for maximal growth, so that a deficiency of any one nutrient would restrict growth regardless of all others (Figure 16.6). Liebig also proposed that growth was determined by the nutrient in most limiting supply and that when that nutrient was supplied, further growth was controlled by the next most limiting nutrient. This ‘Law of the Minimum’ often holds when a deficiency of one nutrient is appreciably more severe than others as in wheat grown on a soil more severely deficient in N than in Zn and Cu (Table 16.3). Notably, wheat plants did not respond to either Zn or Cu fertilisers in the absence of N fertiliser, but responded strongly to both when N was present.

In other situations, plants deficient in two nutrients respond to additions of either nutrient when added alone, but when added together each modifies the other’s response according to the Law of Limiting Factors as stated by Blackman in 1905: ‘the effect of a factor is least when another factor limits growth and greatest when all other factors are in optimal supply’. Plants growing in soils severely deficient in both N and P frequently respond in this way to addition of N and P fertilisers (Table 16.4). A positive interaction between N and P on shoot dry mass is obvious in this case, and discussed further in Section 16.3.5 with N, P and K interactions on growth of oats.

Soils with multiple nutrient deficiencies are common in Australia, so that it is important to recognise this principle of key limiting factors by supplying all other limiting nutrients when testing for a specific nutrient deficiency (Figure 16.6; Section 16.3.5).

FEATURE ESSAY 16.2  Sodium in C4 photosynthesis

Peter Brownell 

My interest in the role of sodium in plant metabolism began in 1954 when my PhD supervisor, Professor J.G. Wood, suggested a project to determine if sodium was required by higher plants. In 1955, Allen and Arnon, at the University of California, Berkeley, demonstrated sodium to be specifically required by the Cyanobacterium, Anabaena cylindrica. Two years later, Brownell and Wood (1957) demonstrated the essentiality for sodium as a micronutrient for Atriplex vesicaria (bladder saltbush). Subsequently, Brownell and Crossland (1972, 1974) showed that sodium was essential for C₄ and possibly crassulacean acid metabolism (CAM) plants. Research into the function of sodium in plants started in the Botany Department, University of Adelaide, and was continued at Waite Agricultural Research Institute prior to my appointment to James Cook University of North Queensland.

Sodium is one of the most abundant elements on earth, so that plant culture under conditions totally free of sodium is extremely difficult. Confirmation that sodium was an essential element for higher plants required rigorous experimental procedures to exclude sodium salts. Water was twice distilled so that it contained less than 0.0002 mg Na mL^–1. Salts were purified by up to six recrystallisations. As a result, total sodium in full-strength culture solution was less than 0.0016 mg Na mL^–1. This is about one-hundredth of the amount that would have been present if the solution had been made up from unpurified analytical reagents. Even the atmosphere in which plants were grown was filtered to prevent contamination from dust particles. When these techniques were implemented for Atriplex vesicaria, sodium-deficiency symptoms of chlorosis, necrosis and greatly decreased growth were observed. Dry mass of sodium-deficient plants was only one-twentieth that of plants receiving 0.5 mg Na mL^–1.

Drawing on studies with other essential nutrient elements, we presumed that sodium would be required by all higher plants. Surprisingly, of the 30 species examined, which included halophytes, other Chenopods and non-endemic species of Atriplex, the only plants that seemed to require sodium were 10 species of Atriplex endemic to Australia. This outcome was perplexing because differences in sodium requirement could not be correlated with any obvious differences between the species studied. We imagined that most higher plants might require extremely small amounts of sodium but perhaps Australian species of Atriplex were exceptional in requiring larger amounts.

Discovery of the C₄ photosynthetic pathway by Hal Hatch and Roger Slack in 1966 provided a clue: perhaps only plants having the C₄ pathway required sodium! C₄ plants from dif-ferent families were subsequently examined and all responded to small amounts of sodium, as did the Australian species of Atriplex. A CAM plant, Bryophyllum tubiflorum, also responded to sodium when growing in CAM mode but this species did not respond to sodium when growing in C₃ mode (Section 2.1). By implication, sodium requirement was somehow linked to the C₄-fixing system.

Our discovery that C₄ plants required sodium suggested a possible function of this element in C₄ plants: was sodium needed for operation of the C₄ appendage in transporting CO₂ to bundle sheath cells for reduction to carbohydrates? This view was substantiated by our observations that sodium-deficiency symptoms are alleviated in plants grown in atmos-pheres with elevated CO₂ concentrations (Figure 2). Plants supplied with sodium showed little or no response to high CO₂. This result suggested that under conditions of sodium deficiency, transport of CO₂ to bundle sheath cells decreased, thus limiting overall rates of CO₂ assimilation. When atmospheric CO₂ was increased from 330 to 1500 µL L^–1, CO₂ must have then reached bundle sheath cells in sufficient amount by physical diffusion, thus bypassing the C₄-fixing system.

Several possible sites were identified where sodium deficiency might limit the rate of supply of CO₂ to bundle sheath cells in normal air (Figure 3). Processes examined, including stomatal conductance (A), carbonic anhydrase (which hydrates CO₂ to form bicarbonate, the substrate of phospho-enolpyruvate (PEP) carboxylase, B), and the activity of PEP carboxylase (C) were all unaffected by sodium nutrition. By contrast, leaves of sodium-deficient plants had high levels of alanine and pyruvate and low levels of PEP, the acceptor of CO₂ in C₄ plants, which suggests a block (D) in the conversion of pyruvate to PEP in the mesophyll chloroplasts.

Regeneration of PEP from pyruvate involves (1) transport of pyruvate into the mesophyll chloroplast, (2) enzymatic conversion of pyruvate to PEP within the stroma and (3) provision of energy for the conversion reaction.

Sodium was not found to affect the activity of pyruvate phosphate dikinase (the enzyme catalysing the conversion of pyruvate to PEP), but in 1987 Ohnishi and Kanai discovered a sodium-induced uptake of pyruvate into mesophyll chloro-plasts of Panicum miliaceum. This immediately suggested a role for sodium in a majority of C₄ plants. Ohnishi and Kanai demonstrated sodium-induced pyruvate uptake in the mesophyll chloroplasts of many other C₄ plants with the exception of some species from Andropogoneae and Arundinelleae. These exceptions have now been shown to have proton instead of sodium ion induced pyruvate transport into the mesophyll chloroplasts.

Mark Johnston and Chris Grof then obtained evidence, in sodium-deficient C₄ plants, for damage to the light-harvesting photosystem which is the source of energy for pyruvate transport and/or the regeneration of PEP. In sodium-deficient plants, they found lower chlorophyll a/b and fluorescence ratios and lowered photosystem II activity, with altered ultra-structure in the mesophyll chloroplasts. Notwithstanding these extra dimensions to sodium effects, a difficult question remains as to the primary function of sodium. Is sodium needed to maintain the light-harvesting and energy-transducing systems in the mesophyll chloro-plasts and/or to transport pyruvate into the mesophyll chloroplasts? If transport, then damage observed in mesophyll chloroplasts in sodium-deficient plants could have been caused by excess energy that would normally have been used to convert pyruvate to PEP. If sodium can be demonstrated to be an essential element for members of Andropogoneae and Arundinelleae, we would conclude that activation of pyruvate transport is not the sole function of sodium in C₄ plants.

Sodium is also involved in transport of metabolites including bicarbonate ions in Cyanobacteria. In 1967, Professor Don Nicholas and I found the activity of nitrate reductase to be many times greater in sodium-deficient compared to normal cells of Anabaena cylindrica. We were unable to obtain a similar effect of sodium deficiency on nitrate reductase in C₄ plants. The effect of sodium nutrition on nitrate reductase in A. cylindrica may have been a consequence of some earlier effects of the sodium treatment, perhaps related to inorganic carbon.

Despite our incomplete understanding of sodium nutrition, we can be confident that a lack of sodium will never limit plant growth in nature. However, as sodium has an important role in C₄ photosynthesis, exploring functional roles continues to be an extremely challenging and exciting endeavour.

References

Allen, M.B. and Arnon, D.I. (1955). ‘Studies in the nitrogen-fixing blue-green algae. II. The sodium requirement of Anabaena cylindrica’, Physiologia Plantarum, 8, 653–660.

Brownell, P.F. and Crossland, C.J. (1972). ‘The requirement for sodium as a micronutrient by species having the C₄ dicarboxylic photosynthetic pathway’, Plant Physiology,
49, 794–797.

Brownell, P.F. and Crossland, C.J. (1974). ‘Growth responses to sodium by Bryophyllum tubiflorum under conditions inducing crassulacean acid metabolism’, Plant Physiology, 54, 416–417.

Brownell, P.F. and Wood, J.G. (1957). ‘Sodium as an essential micronutrient element for Atriplex vesicaria (Heward)’, Nature, 179, 635–636.

Hatch, M.D. and Slack, C.R. (1966). ‘Photosynthesis by sugar-cane leaves: a new carboxylation reaction and the pathway of sugar formation’, Biochemical Journal, 101, 103–111.

Ohnishi, J. and Kanai, M. (1987). ‘Na⁺-induced uptake of pyruvate into mesophyll chloroplasts of a C₄ plant, Panicum miliaceum L., Federation of European Biochemical Societies Letters, 219, 347–350.

Further reading

Brownell, P.F. (1979). ‘Sodium as an essential micronutrient element for plants and its possible role in metabolism’, Advances in Botanical Research, 7, 117–224.

Brownell, P.F., Bielig, L.M. and Grof, C.P.L. (1991). ‘Increased carbonic anhydrase activity in leaves of sodium-deficient C₄ plants’, Australian Journal of Plant Physiology, 18, 589–592. 

16.3.4  Diagnosis of deficiencies

Almost all Australian soils are low in one or more available nutrients, sometimes dominating the distribution of native flora and severely limiting crop and pasture production. Diagnosis of nutrient deficiencies in plants and prognosis of likely nutrient deficiencies in a forthcoming growing season are fundamental to agricultural production and ecosystem management.

One standard procedure for defining those nutrients likely to be deficient in a soil for crop growth is to apply fertiliser trials to crops on representative soil types in the field or in pots. Soil analyses are also useful for some nutrients. Visible symptoms often act as useful guides to nutrient deficiencies especially when interpreted in relation to nutrient function and mobility within a plant. They are sometimes sufficiently specific for definitive diagnosis, as, for example, with split seed in Mn-deficient lupins. Visible symptoms have been especially useful in development of Australian agriculture but they can be too general for diagnosis and may be confused with symptoms of other stresses. For example, both frost and Cu deficiency at flowering can produce identical empty heads of wheat at maturity. In the absence of leaf symptoms, an underlying Cu deficiency will not have been detected until too late, and yield will have already been severely depressed.

One convenient method for assessing soil–plant nutrient status is to analyse total nutrient concentrations in dried plant material. Figure 16.5 shows an ideal relationship of nutrient concentration to plant growth for such diagnosis. Nutrient concentrations below a minimum or ‘critical’ concentration indicate that nutrient deficiency is restricting plant growth; concentrations above another ‘critical’ concentration are toxic; concentrations between the two critical values signify adequate to luxury nutrient status.

Nutrient concentrations in whole-plant shoots have often proved unsatisfactory for several reasons including change in critical values with plant age and low mobility of some nutrients from old leaves to growing regions. In extreme cases, nutrient concentration of severely deficient plants may be higher than that of healthy plants, giving a C-shaped curve commonly known as the ‘Piper–Steenbjerg’ effect, after the first scientists to record it (Figure 16.7). Concentrations in specific plant parts of similar physiological age such as most recently expanded leaf blades in cereals generally give more instructive relationships.

Physiological activities, metabolite concentrations, physical measurements such as in vivo chlorophyll a fluorescence for Mn deficiency (Figure 16.3) and leaf absorption or reflectance of light for N deficiency have all been used as the basis of other diagnostic tests. In addition, enzyme activities and con-cen-trations or ratios of all or selected forms of nutrients in living and dead plant parts and expressed sap find application. Given a clear understanding of nutrient function, physiological tools are excellent for diagnosing individual nutrient deficiencies.

From measurements during growth, diagnostic tests have also been used as prognostic tests which predict the likely impact of nutrient deficiencies at harvest. Such predictions need additional information on the ability of soils to supply nutrients and the size of remobilisable reserves within plants. They must also estimate the likely environmental conditions between sampling and harvest, and for this reason are only indicative. Where sufficient data are available, this information has been incorporated into static and dynamic simulation models which predict the likely value of fertiliser applications and hence provide a firm basis for fertiliser recommendations.

(a)  Deficiencies of group 1 nutrients: N, S

As nitrogen and sulphur are essential components of all the structural and metabolic proteins in plants, they influence all processes of growth and metabolism. For both nutrients the first visible symptom of deficiency is the paling of the green colour of leaves due to their low chlorophyll concentration.

Paling develops first in old leaves of N-deficient plants while young and developing leaves remain green. Paling spreads to younger leaves as the deficiency intensifies. This charac-teristic pattern results from an effect of N deficiency in initiating senescence of older leaves and the export from them of metabolites from the breakdown of their proteins and chlorophyll to younger organs.

While the symptoms of sulphur deficiency also result from loss of chlorophyll from leaves, the pattern of the resulting paleness and yellowing vary with N supply. In legumes dependent on symbiotic fixation of their N supply, the symptoms of S deficiency are identical with those of N deficiency in plants without an effective symbiosis. In non-leguminous plants with a low level of N supply, the symptoms of S-deficiency may also resemble those of N deficiency. But in plants with adequate N, the onset of S-deficiency does not induce senescence in old leaves so that they remain green in S-deficient plants while the young leaves become pale and yellow from failure of chlorophyll synthesis.

Paling and yellowing of old leaves occurs in N- and S-deficient plants, and is often associated with development of reddish colours in petioles and leaves resulting from accumu-lation of anthocyanin pigments. Old leaves of S-deficient plants remain green, while young leaves are generally bright yellow rather than reddish.

Sulphur deficiency was not a problem for most Australian crops and pastures while single superphosphate, which contains over 11% S, was the main source of P fertilisers. Following introduction and expanding use of high-P fertilisers with lower S contents, S deficiency has become more important, especially in pastures.

In soils, inorganic S occurs almost entirely as sulphate, which is much less strongly adsorbed than phosphate. Indeed, for many soils in higher rainfall regions, fertiliser S is readily leached so that autumn applications are lost well ahead of the following spring.

Organic S in soils is closely associated with C and N of organic matter at a level of about 10% of the N content. Mineralisation of soil organic matter in cropping generally supplied sufficient S for crop growth unless fertiliser N is applied. Accumulation of soil organic matter by pastures generally exceeds mineralisation, so that S deficiency is more common with pastures than with crops.

(b)  Deficiencies of group 2 nutrients: P, B

Phosphorus

Like N deficiency, P deficiency often enhances anthocyanin production, producing red or purple colours in plant stems, petioles and leaves. P differs from N deficiency in that leaves have a darker green colour due to a high chlorophyll concen-tration which in turn results from P deficiency depressing cell expansion more strongly than chlorophyll production. P deficiency disturbs most metabolic processes and particularly photosynthesis and carbohydrate metabolism.

Ecologists have found P to be the main nutrient limiting growth of several Australian native plant communities and have suggesed that soil P influences community boundaries and floristic composition. Native soil P has also been almost universally deficient for agricultural production, and especially P-fixing soils such as red-brown earths (oxisols in Table 16.1). Black earths of southeastern Queensland and northern New South Wales (vertisols in Table 16.1) are a notable exception to this chronic P deficiency. Crops have been grown there for over 90 years without P fertilisers, and in small areas of soils in a few other localities P has been adequate.

Many million tonnes of phosphatic fertilisers have now been put on Australian crops and pastures. In all soils except siliceous sands from which it leaches, fertiliser P reacts strongly with Ca, Fe and Al minerals (P sorption in Table 16.1), making a large proportion unavailable to plants. Organic P compounds which accumulate under pastures in most soils tie up additional fertiliser P, and until such soil reactions reach equilibrium P has to be added in excess of immediate plant requirements to ensure maximum plant yield. With continued application of excess P fertiliser, plant-available P in soils eventually reaches a level where maximum yield can be maintained by dressings equal to the amount removed in agricultural produce. Pastures on many soils need around 150–200 kg P ha^–1 to reach that point. Cereals on similar soils need 200–250 kg P ha^–1 to offset losses due to mixing of fertiliser P through a larger volume of soil compared with surface application to pastures. Such applications represent a substantial cost to farmers, and crops on about half of Australia’s agricultural soils remain undernourished with respect to P nutrition.

In terms of soil–plant dynamics, fertiliser P that appears to be rendered unavailable by soil reactions is in fact still acces-sible to plants, but in a protracted fashion. Native ecosystems that operate at a slow tempo have adapted to this inertial release via specialised roots and associations with microorganisms (Sections 3.6, 16.4), but in agricultural environments, soil P is released too slowly to meet internal P requirement for maximum yield of present-day crop and pasture plants. A major challenge in this area for plant physiologists, breeders and genetic engineers is to develop plant cultivars with greater efficiency in extracting P from soils. Such cultivars would lower requirements for fertiliser P by enhancing access to ‘unavailable’ soil P reserves.

Boron

B deficiency symptoms sometimes appear as an interveinal chlorosis of young or recently matured leaves which may be confused with symptoms of other deficiencies. Other symptoms are sufficiently characteristic to provide a strong indication and even a definitive diagnosis of the deficiency. Charac-teristic symptoms include death and discolouration of apical meristems with consequent thickening of stems and multiple shooting of axillary buds; thickening of roots with multiple lateral apices close to the tip; and death and discolouration of internal tissues as in hollow heart of peanut kernels, heart rot of sugar beet and internal cork of apples.

Analogous to symptoms of B deficiency, symptoms of B toxicity also commonly occur as an interveinal chlorosis, but they develop first on old leaves and the pattern of chlorotic areas varies widely with plant species, coinciding with the vein endings in the leaf where B accumulates to high concentrations (Case study 16.1).

Reproductive development is especially sensitive to B deficiency and there are many reports of depressed seed production in crops and pastures with no visible symptoms of B deficiency in vegetative tissues. In subterranean clover (New South Wales production) poor seed production in legumes and other dicotyledons results from inhibition of flowering, shedding of flowers and fruits and poor seed development. Seeds with low B concentrations may also fail to germinate or produce malformed seedlings. In cereals and other monocotyledons, B deficiency depresses seed production by lowering pollen viability and growth.

Many of the characteristic symptoms of B deficiency may be related to some requirement for B in cell wall for-mation. Most B in cells on a marginal B supply is tightly bonded to cell walls, and differences in cell wall composition explain a much higher internal B requirement by dicotyledons compared to monocotyledons.

B deficiency has affected crop production in many countries and especially across South Asia, although susceptibility to B deficiency varies widely among cereals and legumes. B deficiency has not been a widespread problem of crops and pastures in Australasia, but B toxicity occurs in cereals in South and Western Australia on soils formed from parent material of marine origin. Genetic variation in cereals with respect to B absorption has allowed breeding and selection for cultivars tolerant to high B soils (Case study 16.1).

(c)  Deficiencies of group 3 nutrients: K, Ca, Mg, Na, Cl

Potassium

Plant K is present in solution and adsorbed into cellular com-ponents as a univalent cation. K activates more than 50 enzymes and has major roles in membrane transport in osmotic and pH balance of cell cytoplasm and vacuole. K is transported into and out of guard cells as a counter-ion for malate, in the process controlling the opening and closing of stomata (Section 15.2). K is highly mobile within plants, and following onset of deficiency K moves rapidly from older leaves to young organs. Symptoms of K deficiency generally appear first on recently matured leaves and include a general bronzing discolouration or an interveinal chlorosis followed by necrosis described as ‘scorching’. Symptoms of K deficiency on legumes are often sufficiently specific for reliable diagnosis, namely a characteristic pattern of white spots that develops on mature leaves and becomes necrotic, resembling insect damage.

Plants require K in large amounts, second only to N. In some species such as sugar beet and others with halophytic relatives, Na can substitute for K in large part, probably by meeting osmotic functions normally attributed to K. Other species exist where Na/K substitution never occurs, and between these two extremes species and even cultivars vary widely in their Na/K flexibility.

With the exception of areas of light sands, which are especially widespread in Western Australia, most Australian soils contain adequate K for plant growth (clay soils derived from illite are especially well provided with reserves). In the sandy soils mentioned above, K is adsorbed onto organic matter as an easily exchangeable univalent cation which is readily leached. On such soils, development of a soil horizon to trap leached K within reach of plant roots assumes especial importance. Large amounts of K are also leached from living leaves and contribute to K mobility within ecosystems.

K content of actively metabolising cells is high (2–5% dry mass) but declines rapidly with senescence. As a result, removal of green plant material as hay also removes large amounts of K which in turn can induce K deficiency on soils with low reserves.

Sodium and chlorine

Deficiencies of these nutrients have only been seen in plants grown in highly purified environments; they have not been observed in natural or agricultural ecosystems. By contrast, toxicities of Na and Cl are widespread and cause serious problems in many countries (Section 17.1)

Na may benefit plants in any of three distinct ways: Na may be essential; Na may substitute for K when K supply is low; and Na may enhance growth even when K supply is high. Brownell and his colleagues at the universities of Adelaide and then at James Cook in Townsville have established the essentiality of trace amounts of Na for C₄ plants (Feature essay 16.2).

Calcium and magnesium

Deficiencies of Mg are rare in Australia but have been reported for fruit and vegetable crops in New South Wales and Western Australia; citrus trees are especially sensitive. In some cases the deficiency has been induced by heavy appli-cations of K or ammonium fertilisers to acid soils. Mobility of Mg from leaves is intermediate between that of the highly mobile K and immobile Ca and is greatly accelerated by senescence. Mg deficiency symptoms generally appear first in older leaves as interveinal chlorosis but may appear first in young leaves depending upon the rate of development of the deficiency and other environmental factors. Chlorosis results from the breakdown of chlorophyll which has Mg covalently bound in its centre. Each molecule of chlorophyll carries one Mg atom, but in terms of the total pool of leaf Mg, the amount bound to chlorophyll varies from around 35% of total leaf Mg in deficient leaves to 5% in leaves with more than adequate Mg. Another 5–10% of leaf Mg is bound in the cell walls. Remaining Mg is largely ionic and functions as an osmoticum and as a catalyst in a wide range of reactions including phosphorylations and carboxylations.

Ca deficiencies are more common than Mg deficiencies, but are usually restricted to small areas of acid soils or occur sporadically as physiological disorders in particular crops. On acid peaty sands in Western Australia symptoms of Ca de-ficiency have been observed in clover, and were accompanied by symptoms of N deficiency due to low Ca inhibition of N₂ fixation. Application of calcium carbonate corrected both deficiences. Calcium carbonate, despite being only sparingly soluble, was more effective than calcium sulphate because it decreases acidity which inhibits Ca absorption. Although Ca deficiency is relatively rare, calcium carbonate has been widely used to correct a variety of other problems associated with soil acidity (Section 16.5).

A number of puzzling physiological disorders have been recognised as being due to Ca deficiency and attributed to the low mobility of Ca in phloem. These include bitter pit in apples, brown heart in compact leafy vegetables such as brussel sprouts and lettuce, empty pods in peanuts and subterranean clover, and blossom-end rot of tomatoes. Because of the low mobility of Ca in phloem, developing organs must obtain their Ca requirements from the xylem sap or directly from external sources. Fruits and leaves receive their Ca supply in the xylem sap and hence are affected by factors limiting their rates of transpiration, especially humidity. Roots and under-ground fruits, such as peanut and subterranean clover pods, receive little or no inflow of xylem sap, so must obtain their Ca from the surrounding soil.

The low phloem mobility of Ca also results in symptoms of Ca deficiency appearing in growing apices and young regions of the shoot and stem. Following exhaustion of Ca supply, symptoms also appear in older organs as a result of later cell expansion or loss of Ca via exchange with other cations in the xylem sap.

(d)  Deficiencies of group 4 nutrients: Mn, Fe, Co, Ni, Cu, Zn, Mo

Manganese

The first symptoms of Mn deficiency in plants frequently resemble those of Fe deficiency (Figure 16.8) with interveinal chlorosis developing in the youngest leaves and often requiring biochemical tests or elemental analysis for definitive diagnosis. Sometimes interveinal chlorosis develops first in mature leaves apparently at variance with low mobility of Mn from these leaves. While interveinal chlorosis is the first visible symptom, photosynthesis may be severely depressed before chlorophyll concentration is affected. Water photolysis (Section 1.1) is catalysed by an Mn metalloenzyme in which Mn undergoes reversible oxidation–reduction. Only one other Mn metalloenzyme is known — MnSOD. But, unlike other nutrients in this group, much of the Mn is present as divalent ions which act as cofactors to over 30 other enzymes catalysing key metabolic events including oxidation–reduction, de-carboxy-lation and hydrolytic reactions.

Mn deficiency also depresses lignin synthesis, especially in roots. Low lignin content of roots is thought to be responsible for the low resistance of Mn-deficient plants to root diseases such as ‘take all’ (Graham and Rovira 1984).

In peas and lupins with little or no vegetative symptoms, Mn deficiency sometimes affects seed production inducing symptoms such as marsh spot in peas and split seed in lupins which are sufficiently specific to diagnose Mn deficiency unambigously.

Mn deficiency is an important problem for crops in many countries, and occurs on acidic soils containing low concen-trations of Mn and on calcareous soils. Plant-available Mn in soils fluctuates widely and rapidly with environmental con-ditions, increasing with acidity, soil water and anaerobiosis, and decreasing with alkalinity, decreasing soil water and aerobiosis. On calcareous soils, such as those of the Cape Yorke Peninsula in South Australia, Mn deficiency is particularly difficult to correct as any Mn fertiliser is rapidly immobilised in the soil. This situation provides a compelling argument for continuing research into genetic factors underlying mechanisms of Mn uptake from soils and especially species differences in acquisition (Table 16.5).

Iron

The first symptoms of Fe deficiency generally appear as chlorosis in the youngest leaves, reflecting both the retention of Fe in older parts and a function of Fe in chlorophyll synthesis (Figure 16.8). In legumes, older leaves may also become pale due to Fe deficiency inhibiting nodule development.

Fe is also involved in many other plant processes as it is an essential component of many enzymes in which it acts either as a bridging agent to substrates, as in the S–Fe protein aconitase, or as a redox agent, as in heme enzymes such as cytochrome and in S–Fe proteins such as ferredoxin and superoxide dismutase.

Fe deficiency is a serious and widespread problem of crops in many countries but is relatively unimportant in Australia. Fe deficiency is common on calcareous soils where its severity has been correlated in non-gramineous plants with the amounts of poorly crystalline and amorphous Fe oxides and in gramineous plants with HCO₃^– concentrations. These different relationships arise from differences between the two plant groups in the response of their roots to Fe deficiency.
In gramineous plants, the onset of Fe deficiency promotes excretion of organic compounds with the capacity to chelate Fe (phytosiderophores) in soluble complexes which may then be absorbed and metabolised. By contrast, Fe deficiency in non-gramineous plants promotes the excretion of hydrogen ions, solubilising soil Fe by acidification of the rhizosphere; high concentrations of HCO₃^– counteract this mechanism by buffering the rhizosphere against acidification.

Cobalt

Co is required by legumes for N₂ fixation but not for growth of plants given adequate fixed N. Within legume nodules, Co is chelated to N atoms in the centre of the porphyrin structure of cobalamin, which is an essential coenzyme for several enzymes involved in the metabolism of bacteroids and free-living Rhizobium and other N₂-fixing organisms. In legumes the symptoms of Co deficiency are those of N deficiency from delayed nodulation or impaired fixation once nodulated.

Co deficiency in plants is rare in the field but has been demonstrated in subterranean clover on impoverished siliceous sands in South Australia and Western Australia and in lupins on lateritic sandy soils in Western Australia. The amount of Co required is very small and seeds may contain sufficient for the current crop; indeed, the first appreciable field response of lupins to Co was obtained in a crop sown with seed saved from two previous generations on the same soil!

By contrast, Co deficiency in sheep and cattle has been a serious and widespread problem over appreciable areas of New Zealand, southeastern South Australia, and southern Western Australia. Milder deficiencies occur in many other countries.

Nickel

During the 1990s and some years after plant urease was shown to be an Ni metalloprotein, Ni was shown to be essential for legumes and cereals. One of its functions probably involves detoxification of urea. So far, no unequivocal demonstration of Ni deficiency has been shown in field crops although Ni toxicity occurs on both natural and polluted soils.

Copper

Cu deficiency has been a widespread and serious problem in crops, pastures and animals in many countries, including Australia. In plants, Cu is an essential component of many enzymes and non-enzyme proteins involved in electron transfer in many processes including photosynthesis, respiration and lignification. Cu is also required for N₂ fixation in legumes, but the amounts required are very close to those for growth so that, while symptoms of Cu deficiency may appear as N deficiency, they often develop other characteristics as the deficiency becomes more severe. Except where N₂ fixation is involved, symptoms appear first in young leaves and meristems owing to the low mobility of Cu from older leaves before they senesce.

Symptoms often appear as a distortion of young leaves and stems, death of apical meristems with multiple bud develop-ment, leaves with interveinal chlorosis (Figure 16.8) or which are blue-green in colour, and appearance of wilting with flaccid leaves and pendulous branches. Wilting and twisting and distortion of leaves, as characterises Cu deficiency in many species, may be ascribed to poor lignification related to the low activities of two Cu enzymes involved in the synthesis of lignin — polyphenol oxidase and diamine oxidase. Lignin synthesis is sensitive to mild Cu deficiency so that a simple lignin staining test on cut stems may be used to diagnose Cu deficiency in plants.

As with Mn deficiency, poor lignification of roots may account for the susceptibility of Cu-deficient plants to root diseases such as ‘take all’. Poor lignification of anthers in Cu-deficient plants also leads to their failure to open and release their pollen. In addition, pollen from Cu-deficient plants may be sterile. As a result, seed production is particularly suscep-tible to Cu deficiency and wheat crops with no leaf deficiency symptoms have been found at harvest to have set few seed.

Zinc

The most characteristic symptom of Zn deficiency in dicotyledonous plants is the appearance of a rosette of little, misshapen leaves at the stem apex leading to descriptions of the disorder as ‘rosetting’ and ‘little leaf’. Zn deficiency is usually accompanied or preceded by an interveinal chlorosis and necrosis of older leaves and sometimes also by their discolouration from accumulation of anthocyanins. In cereals and other monocotyledons only the symptoms on the older leaves are obvious.

Misshapen leaves and a failure of stem internodes to elongate in rosetting led to the hypothesis over 50 years ago that Zn was required for synthesis of indole acetic acid (IAA). IAA concentrations are depressed in apices of Zn-deficient plants and recover quickly on addition of Zn, but a specific role for Zn in its synthesis has yet to be defined. Meanwhile, Zn has been shown to be a component of many metalloenzymes, to be essential for ribosome structure and integrity of membranes, and as Zn²⁺ to activate enzyme systems. Of the many Zn metalloenzymes, one is involved in gene expression through its binding to DNA, affecting its replication and transcription. This function, together with roles in preserving the structural integrity of ribosomes and in depressing RNase activity, gives Zn a key role in protein metabolism and creates a high requirement for Zn at sites of protein synthesis. As a result, meristematic tissues accumulate high concentrations of Zn; young leaves also have much higher internal Zn requirements than older leaves so that critical Zn concentrations for diagnosis of Zn deficiency vary widely with leaf age.

Zn deficiency has been a widespread problem in many countries, occurring in both acid soils with parent materials containing low Zn and calcareous soils where low pH makes Zn unavailable. Zn has been a particularly important deficiency in southern and western Australia, severely depressing both legume and cereal production. The deficiency would have been even more severe and widespread had Zn not been a contaminant of many macronutrient fertilisers including superphosphate. With replacement of superphosphate by P fertilisers with lower Zn contents, crops in soils considered from previous experience to have adequate Zn now show Zn deficiency.

Molybdenum

Symptoms of Mo deficiency and plant requirements vary with species and environmental conditions. In legumes and other N₂-fixing plants on soils low in N, Mo deficiency is expressed as an N deficiency due to failure of N₂ fixation by a symbiotic system that requires Mo for nitrogenase. In all plants supplied with adequate N as nitrate, leaves may become pale, malformed and necrotic as N metabolism fails and nitrate accumulates to toxic levels due to low activity of the Mo metalloenzyme nitrate reductase. Plants with adequate nitrate supply need much less Mo than plants fixing N₂. Plants supplied with N as ammonium under sterile conditions require only very small amounts to forestall symptoms.

Variation in expression of Mo deficiency in crops and pastures reflects differences in Mo requirements for particular functions such as symbiotic N₂ fixation. Mo deficiency was thus a widespread problem in pastures where legumes were sown to improve N status of low N soils (Figure 2 in Feature essay 16.1). Mo is particularly important for legume-based pastures on acidic soils in eastern Australia. Responses of non-leguminous crops to Mo are much more restricted.

16.3.5  Nutrient interactions

As outlined above, agricultural production on Australia’s impoverished soils has been severely limited by widespread deficiencies of various nutrients. Twin deficiencies of N and P were ubiquitous and often accompanied by one or more additional deficiencies, so that nutrient interactions on plant growth and crop productivity have assumed unusual significance. P was the first deficiency to be recognised, and after 1882, P fertilisers were used increasingly on wheat. Years later, N deficiency rather than water deficiency was appreciated as a major restriction on crop response to added P. N fertilisers proved too expensive for wheat production, and interest focused on legume pasture leys to build up soil N reserves.

In pastures, N × P responses affect botanical composition (Section 12.3.3) as well as dry matter production. H.C. Trumble and co-workers clarified underlying principles in sand culture experiments at the Waite Institute in Adelaide with subterranean clover and rye grass. When established separately, rye grass only grew well when both N and P were added, while clover responded strongly to P irrespective of added N. When grown with clover, rye grass had little effect on clover in the absence of added N, but rye grass suppressed clover when N was added. These findings explained field observations on changes in grass and clover components in response to fertiliser P and fluctuations in soil N.

In many areas of Australia, introduction of legumes and their special requirements for Rhizobium sp. plus nutrients for N₂ fixation brought a host of new interacting factors and problems. In resolving these problems, A.J. Anderson and his colleagues at CSIRO in Adelaide and Canberra developed and applied innovative ways of interpreting fertiliser responses and their interactive effects on plant growth and reproductive development (Anderson 1956). They replotted Mitscherlich’s early and extensive data on oat response to N, P and K (Figure 16.9), observing for these response surfaces that any two nutrients occurring in a 2 × 2 factorial arrangement frequently showed a positive interaction. Gross imbalance between N, P or K additions did result in mild toxicity and growth was depressed by excess N, P or K, and especially when applied in combination with minimum levels of accompanying nutrients (see supraoptimal effects in Figure 16.9).

Anderson and co-workers contrasted these interactions between two independently essential nutrients with the negative Mo × N interaction they obtained with clover. In that case, clover showed a strong response to addition of Mo in the absence of N; but clover response to added Mo tended to disappear as more N was added (Figure 16.10a). A negative Mo × N interaction of this sort would be expected if Mo was concerned solely with symbiotic N fixation. By contrast, a plant such as flax (Figure 16.10b) is totally dependent on existing soil N, and showed only a mild additive response to N + Mo compared to N alone.

Anderson (1942) used the same approach to solve other problems of legume establishment including a negative Mo × lime interaction and a negative lime × Rhizobium interaction to show that responses to many tonnes of lime could be re-placed by a few grams of Mo plus small applications of lime and Rhizobium with clover seed at sowing. Large areas of southern Australia previously thought too dry and inhospitable for improvement were subsequently sown to legume-based pastures as a direct result of these research outcomes.

16.4.1  Symbiotic associations

Plants in nature always grow amidst soil microorganisms, and some become intimately associated with plants to form mutualistic symbioses. Examples of such symbiotic micro-organisms include mycorrhizal fungi of various types and N₂-fixing prokaryotes, especially rhizobia. Looser symbiotic associations involve bacteria and soil microfauna within the rhizosphere. Their metabolic activities increase nutrient availability (Sections 3.4, 3.5).

All of these symbioses may affect rates of growth and eventually reproduction of plants compared with growth in the absence of such associations. A symbiotic association is therefore a potential selection pressure that can influence the evolutionary success of vascular plants and hence the com-position of plant communities.

(a)  Diversity and ecology of mycorrhizal plants

Mycorrhizas (sometimes called mycorrhizae or mycorrhizal associations) are by far the most common mutualistic symbioses involving vascular plants, and about 90% of higher plant families (both gymnosperms and angiosperms) contain species that can form mycorrhizal associations. Roots of most ferns also form mycorrhizas and similar structures occur in the absorbing organs of many bryophytes (Harley and Smith 1983; Smith and Read 1997). Fossil mycorrhizas occur in rocks formed in the Devonian era, about 400 million years ago, and present-day mycorrhizas are the result of a very long period of worldwide co-evolution between plants and soil fungi.

Mycorrhizas fall into distinct classes, depending on the types of fungi and plant, and on the anatomy of the mycorrhizas themselves (Section 3.4). Each main class shows wide specificity where a given species of mycorrhizal fungus can colonise different host species and one plant species can be colonised by different fungal species. This situation contrasts sharply with the extremely narrow specificity of N₂-fixing symbioses (Section 3.5).

There are broad differences between the dominant classes of mycorrhizas that are found in different ecosystems, depending on vegetation type, soil characteristics and growth-limiting nutrients. A walk up a mountain can pass through different mycorrhizal ‘zones’ (Figure 16.11). Ectomycorrhizas (with their distinct external fungal sheath and with fungi penetrating intercellular spaces of the root cortex) occur on forest trees including eucalypts, beech (Fagus and Nothofagus), birch, oak and most conifers such as pine and spruce. Ericoid mycorrhizas (where fungi penetrate root cells) occur in plants of heathlands. They are also common in plants growing on peaty soils (those containing large quantities of organic matter) as on the Siberian tundra.

Overall, the most common mycorrhizal associations are vesicular-arbuscular (VA) mycorrhizas, where fungi again penetrate root cells and form finely branched arbuscules within those cells. Fungal storage bodies (vesicles) can form as either intercellular or intracellular structures. VA mycorrhizas predominate in understorey plants of woods and forests of all types, in temperate and tropical grasslands, in plants of scrub and desert and in many tropical forests.

Some classes of mycorrhizal fungi can grow in soil in the absence of a host plant and in pure culture, but VA mycorrhizal fungi cannot. A vast but unsuccessful research effort has gone into attempts to culture these fungi. Instant fame will be accorded whoever discovers the right medium!

The broad ecological zonations for different classes of mycorrhizas should not be overemphasised because the different classes can often be found very close together. For example, many trees of tropical rainforests form VA mycorrhizas but there are exceptions. Dipterocarps, which dominate many tropical rainforests, form ectomycorrhizas, while the other plants in the same forest will nearly all have VA mycorrhizas. Mediterranean-type vegetation — which is quite common worldwide — contains very mixed mycorrhizal associations, including ericoid, ecto- and VA mycorrhizas. Only in very inhospitable soils (e.g. saline, anaerobic and highly disturbed soils and those in very cold environments) are plants mostly non-mycorrhizal.

Since mycorrhizal symbioses are so common, research into the growth and nutrition of plants in the field should always take into account their probable occurrence as a
factor that can influence plant growth and competition, especially in soils where levels of essential inorganic nutrients are low. For example, research done in Western Australia has shown that disturbance of soils during surface mining can reduce the levels of mycorrhizal fungi, but survival in stored topsoil is still sufficient to help re-establish native vegetation when the topsoil is replaced after mining operations cease (Jasper 1994).

Mycorrhizas can also improve productivity in cultivated plants even when levels of applied fertilisers are high. An important example in parts of Australia is the ‘long fallow disorder’ that reduces the growth of a wide range of crops when soils are left fallow for long periods (Table 16.6). This effect with sorghum and sunflower grown in Queensland (Thompson 1987) is due to a massive decline in populations of VA mycorrhizal fungi. Better growth of both plants on soils left fallow for shorter periods was associated with greater colonisation of roots by VA mycorrhizal fungi. There were higher levels of P per plant grown after short fallow and in sunflower P was higher even when expressed on the basis of % of dry weight; these effects reflected uptake of soil P via mycorrhizal fungi.

Improved growth of forest trees after inoculation with specific ectomycorrhizal fungi has been demonstrated in a wide range of sites, including eucalyptus forests in Australia and elsewhere (Grove and Malajczuk 1994). These examples confirm an extensive interest in establishing and managing populations of mycorrhizal fungi in soils to increase plant productivity in agricultural and forest ecosystems (Robson et al. 1994).

(b)  Benefits and costs of symbioses

Physiological benefits to a mycorrhizal plant include improved absorption of nutrients such as P and Zn via external fungal hyphae. Plant–water relations might also be improved, which would be important in arid conditions, but this is still a controversial area of research (Smith and Read 1997). Mycorrhizal fungi do not fix atmospheric N₂ but can facilitate uptake of both inorganic and organic N and can broaden the range of sources of soil N that are available to plants (Case study 16.3). As shown in Queensland, mycorrhizal seedlings of Eucalyptus species can utilise sources of organic N that cannot be assimilated by non-mycorrhizal seedlings (Turnbull et al. 1995). In addition, external hyphae of mycorrhizal fungi can link plants of the same or (within limits) different species and can allow transfer of inorganic and organic compounds between plants.

The idea that mycorrhizal plants can form linked and mutually beneficial communities is an attractive one but it is not yet established if the amounts of solutes transferred from donor to recipient plants are always physiologically important. Transfer of organic C between linked plants is greatest when the recipient is shaded so that its photosynthesis is reduced. Accordingly, transfer of carbohydrate from parent plants might well assist seedlings to grow in deep shade as on a forest floor before they become exposed to bright sunlight (Smith and Read 1997; Smith and Smith 1996).

Mycorrhizas in heterotrophic angiosperms such as some orchids lacking chlorophyll are physiologically distinctive. Even when mature, the plant drains the fungus of organic C derived from other sources (such as soil or donor plants that are autotrophic). This type of mycorrhizal fungus can also supply inorganic nutrients to the plant, but whether the fungi gain any nutritional benefit remains to be established, so that such symbioses may not be truly mutualistic.

Where mycorrhizal fungi colonise autotrophic (photosynthetic) plants the fungus derives its organic C from the plant (Figure 3.17). This is a physiological cost to the plant but one which is usually not excessive, since mycorrhizal plants often grow faster than non-mycorrhizal plants of the same species under the same conditions. These positive growth effects are largest in shoots. Mycorrhizal plants sometimes grow root systems no larger or even smaller than those in non-mycorrhizal plants, diverting organic C to the fungus. However, there are some physiological conditions in which total growth of mycorrhizal plants is the same as or even slower than that of non-mycorrhizal plants of the same type. One of these conditions is low light intensity and here the cause of the effect is obvious: low light means that photoassimilate may be in short supply and the drain to the fungus may be deleterious to the plant.

The second condition resulting in slower growth of mycorrhizal plants is where levels of available soil P are high. The simplest explanation here is that the supply of soil P is adequate even for non-mycorrhizal plants and the C drain to the fungus (where present) then becomes a limitation on plant growth. Under these last conditions some plants reduce or prevent formation of mycorrhizas in a way that is analogous to the absence of N₂ fixation where legumes grow in soils high in nitrate or ammonium.

Finally, some mycorrhizal fungi even ‘cheat’ their hosts by obtaining organic C without supplying P or other inorganic nutrients to their host. Why a plant tolerates being cheated in this way is not known (Smith and Smith 1996). Lack of positive growth responses to colonisation is also sometimes found under a variety of field conditions which are less easy to explain and are the subject of much research.

‘Mycorrhizal responsiveness’ (Table 16.7) summarises the most important factors which influence the extent to which a plant benefits from the symbiosis by improved nutrition and growth. These factors include developmental and anatomical properties of the fungi, including their growth rates in soil, their ability to colonise plants and subsequent growth within roots, and their efficiency in absorbing nutrients from soil and transferring them to the plant. Plants such as some cereals which have long, highly branched roots with long root hairs often show small mycorrhizal responses because the roots themselves are well structured for exploring large volumes of soil. Here, mycorrhizal hyphae confer relatively little extra advantage.

The interface between fungus and plant is obviously crucial in determining mycorrhizal responsiveness, a principle that applies equally to both intercellular and intracellular inter-faces. If there is a sufficiently large area of contact between the symbionts, as in a VA mycorrhiza with many arbuscules, then there is the capacity for operation of a large number of transport proteins that catalyse selective transfer of nutrients. However, the rates of transfer are subject to regulation that can determine the relative amounts of C transferred to the fungus per amounts of P (or other essential nutrients) transferred to the plant and hence the nutritional costs and benefits to the partners.

Table 16.7 also shows effects within plant communities that influence mycorrhizal responsiveness. Positive mycorrhizal growth responses decrease as the density of plants increases and competition for soil nutrients intensifies (Sanders et al. 1995). Put another way, where large numbers of roots and mycorrhizal fungal hyphae are close together, mycorrhizal roots appear to compete with each other and with any non-mycorrhizal roots, so that growth of all the individual plants is reduced compared to growth of plants spaced well apart. This effect is shown in Figure 16.12 and may be one reason why intensive mycorrhizal colonisation fails to benefit field growth.

Hyphal links between plants of the same or different species complicates the issue of competition enormously. If nutrients or organic C are transferred via the hyphae between plants growing under similar conditions, what determines whether a plant is a donor or recipient of resources? This important issue awaits resolution.

Finally, mycorrhizal fungi can suppress infection by some pathogenic organisms — an obvious extra benefit of a symbiotic association. Not surprisingly, responses of field plants are more complicated and difficult to understand than in experiments with potted plants under controlled conditions!

(c)  Selection pressures resulting from mycorrhizal symbioses

A successful mycorrhizal symbiosis can act as a selection pressure. Such symbioses can affect growth and reproduction compared to non-mycorrhizal plants under equivalent conditions. This selection pressure can act within a population of a single plant species if the mycorrhizal fungus is present irregularly in the soil. Consider, for example, the potentially faster growth of an individual plant that becomes mycorrhizal compared to one that cannot or can do so only more slowly. The selection pressure can also act within a community of mixed species. Compare here the potentially faster growth of individuals of a species that forms mycorrhizas compared to individuals of a species that cannot. The subtle and complex nature of the selection pressures arises from the various features that can determine mycorrhizal responsiveness (Table 16.7), which is why ‘potentially’ was emphasised. Nutritional benefits in VA mycorrhizal plants can extend from increased vegetative growth to reproductive effects including earlier flower production, increased production of flower buds and increased seed production. In addition to improving plant fecundity, formation of mycorrhizas can improve the quality of plant offspring, for example their weight and nutrient content, and even the nutrient content of their seeds. These
persistent effects are good evidence that formation of mycorrhizas can bring about strong effects on plant populations and on the structure of plant communities (Sanders et al. 1995).

(d)  ‘Non-mycorrhizal’ species

Some terrestrial plants never form mycorrhizas even under growth conditions where colonisation would be expected. These plants belong to diverse families that are not closely related but include many chenopods and most brassicas (unfortunately including Arabidopsis). There are also individual ‘non-mycorrhizal’ genera within families that otherwise form mycorrhizas. One good example is the legume Lupinus (lupin). In such cases, an absence of these symbioses cannot be construed as disadvantage in evolutionary terms, so that mycorrhizal associations are not necessarily a universal advantage to terrestrial plants. A different range of adaptive features allows these plants to grow and reproduce successfully in the absence of mycorrhizas. These include excretion of organic acids that aid solubilisation of P and production of long, relatively fine roots with long root hairs, which exploit soil P and other immobile nutrients in much the same way as do the external hyphae of mycorrhizal fungi, where present. (Such root properties often reduce mycorrhizal responsiveness as mentioned above.)

Notwithstanding alternative adaptations, an important question remains. What prevents colonisation by mycorrhizal fungi in non-mycorrhizal species? The answer is probably not the same for the different families and is still not known for many plants. In some cases, including brassicas, there is production of chemical defences against colonisation by mycorrhizal fungi. This response has similarities to chemical defence reactions in plant roots against pathogenic organisms (Smith and Read 1997).

(e)  Concluding remarks

When considering effects of mycorrhizas on plant growth and the selective advantages of those associations, all levels of organisation from molecular to ecological are relevant.

A molecular aspect as yet unknown is how plants distinguish between a mycorrhizal fungus that is usually beneficial and a fungus which is harmful (pathogenic). Pathogenic organisms can elicit expression of chemical defences by resistant plants, but what exactly is the sequence of recognition signals for a symbiotic association that results in such chemical defence reactions being turned off? Research into this aspect of mycorrhizal associations lags behind work on N₂-fixing symbioses.

At an ecological level, mycorrhizal fungi also have beneficial effects on important soil properties — they improve soil structure by binding together soil particles to form water-stable aggregates, and in so doing contribute further to the sustainability of managed ecosystems in agriculture, forestry and horticulture.

CASE STUDY 16.3  Rainforest succession and nitrogen nutrition

M.H. Turnbull

Nitrogen (N) is a key limiting nutrient in natural ecosystems, partly because plants have a high requirement, but mostly because readily available forms are often in short supply (Case study 16.2). Foliage commonly contains high concentrations of N (1–5% of dry mass) and meeting this need is a major energy-requiring process because N uptake by plants and N assimilation within plants are both metabolically expensive (Chapin et al. 1987).

Energy requirement is especially high if nitrate is the sole source of N because biological reduction of nitrate ions and subsequent incorporation into plant N metabolism has a higher requirement for both ATP and reductant than does direct use of ammonium compounds. Much of this energy comes via energy transduction from sunlight, and under light conditions which limit growth (e.g. forest understorey) nitrate assimilation in leaves must compete with other synthetic events for biological forms of energy derived from chloroplasts.

In high light environments, excess ATP and reductant from photosynthetic energy transduction (Section 1.2) can be used in leaves for photoreduction of the nitrate that reaches transpiring leaves via xylem flow. Light-dependent reduction of nitrate in leaves is less costly in energy terms than nitrate reduction in roots (Raven 1985), so that some selective advantage is derived from a leaf-based system for nitrate reduction provided solar radiation is abundant.

Accordingly, pioneer and gap species that are sunloving (Section 12.1.3) express levels of nitrate reductase activity that are 5–20 times those of understorey species in both Australian rainforests (Stewart et al. 1988) and Mexican communities (Figure 1; Fredeen and Field 1992).

Nitrate reductase activity of pioneer species is located primarily in leaves, and in line with sun/shade adaptation this prime site appears to be genetically set rather than environmentally driven. Leaf activity is none the less modulated by light because nitrate reductase activity in shoots becomes even more pronounced compared with roots with increased photon irradiance, and especially in conditions where photosynthesis is light saturated. By implication, nitrate reduction is an energy-effective mechanism for N assimilation by sunloving plants in strong light on open areas or in major disturbance gaps of forests.

As a consequence of their high leaf nitrate reductase activities, early-successional (pioneer) species are well placed to take advantage of soil nitrate from disturbed sites such as large gaps or along forest edges. Warm soils in those places could also favour nitrification via temperature-driven microbial activity. An important implication of the very low nitrate reductase activities in species of later-successional stages of forest development is that these species are likely to be using a source of N other than nitrate such as ammonium and complex organic N compounds.

One further advantage of a nutritional strategy involving leaf nitrate reduction at high photon irradiance in pioneer/gap species comes from dissipation of excess photochemical energy that might otherwise contribute to photoinhibition (Smirnoff and Stewart 1985; Section 12.1.2).

Mycorrhizas and plant access to organic nitrogen

Nitrate N is widely recognised as a key source for sunloving (early-successional) plants on disturbed sites, but soil nitrate is less abundant during later-successional stages of rainforest dynamics or in long-established and undisturbed communities. However, complex organic forms of N together with unmineralised ammonium compounds do abound, raising a question as to their availability for plant growth. Some plants are long known to show a distinct preference for ammonium over nitrate ions as an N source; less known is the ability of mycorrhizal associations to extend the nutrient base of their host plants even further to include organic forms of N.

Mycorrhizal fungi feature prominently in the P nutrition of their host plants, and especially on impoverished sites (Sections 5.3.4 and 16.4.1) but their significance for N nutrition is less clear. In growth experiments with seedlings of Eucalyptus spp. (Botany Department, University of Queensland) non-mycorrhizal plants in sterile culture grew poorly on organic N. By contrast, ectomycorrhizal infection enabled seedlings to use organic sources of N and they grew much faster. Infected plants grew well on a range of amino acids and even on protein N (Figure 2). This capacity to use a broad spectrum of organic sources has important implications for tree seedling nutrition. In field situations, a number of N sources might thus be available despite their low concentrations. This presumed ability to access a diversity of N sources might confer distinct nutritional advantages on mycorrhizal plants in forest ecosystems.

When mycorrhizal seedlings of Eucalyptus grandis were offered a mixed source of N in the growth medium, they derived 50% of total N from the amino acid asparagine and 40% from albumen protein (Turnbull et al. 1996). Organic sources of N may thus be significant to mycorrhizal plants even in the presence of readily assimilable inorganic ions, especially where plants must scavenge for sparse resources. However, it would be misleading to suggest that only those plants with a fungal compatibility are able to utilise organic sources of nitrogen directly. We now know that obligately non-mycorrhizal species such as Hakea sp. from coastal heathland in Australia (Turnbull et al. 1996) and Eriophorum vaginatum from arctic tundra (Chapin et al. 1993) can also incorporate labelled amino acid sources. Clearly, some plants are able to short-circuit the mineralisation cycle commonly viewed as the sole source of usable N, either directly or via symbiotic association.

Taken overall, flexibility in N nutrition ensures that a selective advantage during colonisation on a disturbed site where nitrate N predominates is not lost as forests mature and nitrate N is replaced by ammonium N and more complex forms of organic N.

References

Chapin, F.S., III, Bloom, A.J., Field, C.B. and Waring, R.H. (1987). ‘Plant responses to multiple environmental factors’, BioScience, 37, 49–57.

Chapin F.S, III, Moilanen, L. and Kielland, K. (1993). ‘Preferential use of organic nitrogen for growth by a non-mycorrhizal arctic sedge’, Nature, 361, 150–153.

Fredeen, A.L. and Field, C.B. (1992). ‘Ammonium and nitrate uptake in gap, generalist, and understorey species of the genus Piper’, Oecologia, 92, 207–214.

Fredeen, A.L. and Field, C.B. (1996) ‘Ecophysiological constraints on the distribution of Piper species’, in Tropical Forest Plant Ecophysiology, eds S.S. Mulkey, R.L. Chazdon and A.P. Smith, 597–618, Chapman and Hall: New York.

Raven, J.A. (1985). ‘Regulation of pH and osmolarity generation in vascular land plants: costs and benefits in relation to efficiency of use of water, energy and nitrogen’, New Phytologist, 101, 25–77.

Smirnoff, N. and Stewart, G.R. (1985). ‘Nitrate assimilation and translocation by higher plants: comparative physiology and ecological consequences’, Physiologia Plantarum, 64, 133–140.

Stewart, G.R., Hegarty, E. and Specht, R.L. (1988). ‘Inorganic nitrogen assimilation in plants of Australian rainforest communities’, Physiologia Plantarum, 74, 26–33.

Turnbull, M.H., Goodall, R. and Stewart, G.R. (1995). ‘The impact of mycorrhizal colonisation upon nitrogen source acquisition and metabolism in seedlings of Eucalyptus grandis Hill ex Maiden and Eucalyptus maculata Hook’, Plant, Cell and Environment, 18, 1386–1394.

Turnbull, M.H., Schmidt, S., Erskine, P.D., Richards, S. and Stewart, G.R. (1996). ‘Root adaptation and nitrogen source acquisition in natural ecosystems’, Tree Physiology, 16, 941–948.

16.4.2  Parasitic plants and carnivorous plants

Many families of vascular plants have representatives which exhibit either parasitism and carnivory and, given the wide taxonomic separation between such species, various forms of these remark-able adaptive features must have evolved independently, and on numerous occasions over their evolutionary history.

Two examples will be considered. The first concerns mistletoes (Amyema spp.), hemiparasitic flowering plants which acquire water, nutrients and certain organic compounds by tapping the xylem stream of branches of their tree hosts by means of specialised feeding organs (haustoria; Figure 16.13A). The second concerns carnivorous plants (Drosera spp.; Figure 16.13B) which trap insects with mucilage-producing glands on their leaves. The same glandular tissue that secretes mucilage then produces extracellular hydrolytic enzymes (proteases, peptidases and esterases) which digest the protein and other degradable materials of the insect carcasses. The resulting solutes (e.g. amino acids, sugars, phosphorus compounds) are then absorbed. Each case offers distinctive patterns of plant nutrition.

(a)  Mistletoes and host plants

Mistletoes are essentially rootless plants whose mature shoots attach directly to their woody hosts by means of graft-like junctions (Figure 16.13A). Flowering of Australian mistletoes occurs over a long period and their fruits ripen sequentially (Figure 16.13C). Fleshy fruits are eaten and seeds distributed almost entirely by the Australian mistletoe bird (Dicaeum hirundinaceum). Defecating birds wipe the extremely sticky seed masses, only recently ingested, onto adjacent branches of the host tree. Birds obtain only a small nutritional reward from these seeds. They eat large quantities of mistletoe seeds which take less than an hour to pass through. Seeds germinate immediately and, if branch contact is successful, a root-like haustorial structure digests its way into the host’s xylem stream. Once established, a complex ever-enlarging graft union then forms with the host. Secondary growth of the mistletoe haustorium is closely aligned laterally and becomes fully integrated with that of the host branch. Lumen-to-lumen con-tinuities are eventually established at the haustorial interface between tracheary (xylem) conducting elements of the parasite and exposed xylem vessels of the host (Figure 16.13D). It is through these connections that bulk mass flow of xylem vessel fluids takes place from host to parasite. However, most of the haustorial interface consists of parenchyma cells which are apparently involved in selective, metabolically controlled uptake of host nutrients (Figure 16.14).

Growth and survival of xylem-tapping mistletoes depend upon maintenance of a negative gradient in leaf water potential across the haustorial junction between host and parasite. This gradient is maintained day and night in favour of the mistletoe essentially by the mistletoe transpiring faster than its host. This feature, combined with the higher osmotic pressure of mistletoe compared to the host, results in a mistletoe being able to obtain water even when its host is severely water stressed (Case study 15.4).

Rates of photosynthesis of mistletoes are generally less than those of their hosts and this attribute, combined with high transpiration rates, results in the parasite usually exhibiting poorer water use efficiencies than its host (fewer millimoles of CO₂ fixed per mole H₂O transpired). One consequence of this high intake of xylem fluid is that mistletoes become progressively much more enriched than their hosts with respect to certain nutrients, especially K (Figure 16.14). Studies of mineral balance of a mistletoe throughout a season (Pate et al. 1991a) have shown that mistletoes abstract more than sufficient nutrients to meet requirements for growth and reproduction. Running at such an excess, mistletoes drop their leaves when still fully loaded with nutrients, whereas the host is likely to show the reverse of this and engage in most efficient pre-senescence retrieval of key limiting elements such as N, P and K. Mechanisms underlying these very different mineral economies of mistletoe and host have been defined (Figure 16.14).

As a further corollary to fast transpiration and direct intake of xylem fluid, mistletoes derive significant amounts of C in the form of organic acids, sugars, amino acids and other organic solutes absorbed from the xylem of their hosts. This heterotrophic intake of organic solutes may meet from 10% to 62% of a mistletoe’s daily requirement for C, and thereby compensate considerably for their relatively poor photosynthetic performance. Such inputs of C may be especially important where mistletoes are sited in poorly illuminated positions well inside their host tree canopies.

Leaves of Australian mistletoes are generally soft and fleshy in contrast to the tough sclerophyllous foliage of most host tree species. Being potentially attractive sources of food for a range of insects and grazing vertebrates, mistletoes have developed a range of anti-nutritional defence systems rendering them unpalatable or even poisonous to all but a few co-adapted predators. For example, mistletoes of the genera Amyema and Lysiana contain high concentrations of toxic amino compounds such as tyramine and djenkolic acid. In some cases the poisonous compound in question is obtained already synthesised from the host xylem and merely becomes concentrated in tissues of the mistletoe (Pate et al. 1991b). In other cases biosynthesis of the protective compound takes place within the parasite, using raw materials imported from the host.

(b)  Carnivorous plants

Sundews of the genus Drosera are found in every major land mass of the world, but almost three-quarters of the several hundred known species are found in Australia. They typically inhabit nutrient-poor acidic habitats where their ability to capture insects or other arthropod prey confers advantage by supplementing sparse sources of soil nutrients. N supplied by digestion of insect protein is the most important benefit to be derived from a carnivorous habit, especially where a habitat is markedly deficient in N. Insects are, of course, also concentrated sources of other important nutrient elements including P, S and micronutrients. Nevertheless, a carnivorous habit is not obligatory because many insectivorous species grow successfully and reproduce in artificial culture when fed exclusively with inorganic sources of nutrients.

Studies on species of Drosera growing in natural habitats such as the highly impoverished sand plain soils of Western Australia have shown that artificial feeding of leaves with small prey organisms such as spring tails (Collembola sp.) or fruit flies (Drosophila sp.) promotes a measurable improvement in plant growth and reproduction, giving rise to concomitant increases in tissue concentrations of N, P and other key nutrients in plant dry matter (Karlsson and Pate 1992). Growth in nature may therefore be limited by availability of prey.

While proving that benefit does result from this carnivorous habit, a question remains as to how dependent these plants are on insect prey in their natural habitats. A direct way to test this would be to record each item of prey caught by a carnivorous plant over a whole growing season, total up the amount of N in the seasonal catch and then compare this amount of N with the total increment in biomass N made by the plant over the season. A study of this kind, conducted by Dixon et al. (1980), used clonal populations of the southwestern Australian tuberous sundew Drosera erythrorhiza (Figure 16.13E) and included an ¹⁵N-labelling pot culture study to determine the efficiency with which insect N was consumed by leaves. Over three-quarters (76%) of N from the ¹⁵N applied as labelled fruit flies was absorbed. Using this information, and corresponding data on the total N of the catch and of the total N increment of the clones in natural habitat, about one-sixth (16%) of N gained by the clone must have come from native prey.

Such an investigation is not only time consuming but unfortunately provides only one value for dependence on insect N in a single population of only one species in a specific season and at one restricted locality. A much more powerful approach, also applied to southwestern Australian carnivorous plants, is to utilise differences in natural abundance ratios for the two stable isotopes of N (¹⁴N and ¹⁵N; Section 3.5.5). Data for insects caught by a carnivorous species are compared with natural abundance ratios for N in dry matter of that species and in cohabiting non-carnivorous species.

Natural abundance ratios for ¹⁵N:¹⁴N are usually expressed on a relative delta scale (δ¹⁵N as ‰) where a δ value of zero represents the ratio of isotopes found in atmospheric N. Positive δ values for a sample then denote enrichment in ¹⁵N relative to ¹⁴N, negative values the reverse. To explain this application, suppose that N within an insect caught by a carnivorous species showed a δ value of +5‰, while that of the N in dry matter of the carnivorous species was +4‰ and that for non-carnivorous reference species growing purely on soil N in the same habitat was +3‰. Your conclusion, based on simple arithmetic, would then be that the carnivorous species had gained half of its N from insects and the remaining half from available sources of N in the soil. Soil δ¹⁵N value of the latter was assumed to have been monitored effectively using the neighbouring non-carnivorous reference species. Values obtained in such a study (Schulze et al. 1991), encompassing a range of different growth and life forms of southwestern Australian sundews, indicated values for dependence on insect N of approximately 50% across all species studies, but lower values for rosette than for erect and climbing species.

In a special segment of the above study δ¹⁵N values were assessed for (1) normal plants of Drosera erythrorhiza carrying fully glandular leaves and visibly active in catching insects, and (2) certain mutant plants which completely lacked glands and would therefore have been totally dependent on soil N. These mutants provided perfect reference material to compare with normal prey-capturing representatives of the species. The resulting comparison of δ¹⁵N values for three matched adjacent clones of glandular and non-glandular plants indicated proportional dependence on insects of 12.2%, 14.3% and 32.3% (Schulze et al. 1991). These values are in reasonable agreement with the earlier value of 16% for the same species in the prey-inventory study.

Finally, a particularly interesting bug, the hemipteran Cyrtopeltis sp. (Figure 16.13F), lives on a number of species of carnivorous plants native to southwestern Western Australia. This bug has the unusual ability to traverse glandular leaves of a carnivorous plant without becoming caught in that plant’s ‘fly paper’ surfaces. The bugs forage among fresh carcasses on leaves at night, hiding during the daytime in lower non-glandular parts of the shoot. Does such scavenging comprise the major source of nutrition for Cyrtopeltis sp., or, like many other hemipterans, does it feed principally on plant sap? Again, using natural abundance δ¹⁵N values, J.S. Pate and co-workers in Western Australia solved the problem by comparing δ¹⁵N values for freshly collected carcasses, the insectivorous plant (Byblis sp.) on which the insects had been caught, and adults of Cyrtopeltis sp. that were inhabiting the Byblis. Cyrtopeltis sp. showed δ¹⁵N values in the range +4 to +6‰ (i.e. very close to that of the insect prey range 3–6‰) but several δ units more positive than that of the plant (+2 to +3‰). These δ¹⁵N values imply that Cyrtopeltis sp. is a carcass-scavenging non-vegetarian species, and only indirectly harmful to carnivorous plants by robbing them of nutritional substances from their insect catches, rather than drawing directly on plant sap.

16.5.1  Soil acidification

As soils acidify, plant growth generally declines and there is a decrease in the range of plant species that may be grown. The activity of soil fauna and flora decreases also. The extent of acid soils and the degree to which plant growth is reduced by acid soil factors make these soils of considerable ecological and economic importance.

Soil pH (negative logarithm of the molar activity of hydrogen ions) has been used to define acid soils. Since pH is measured on a logarithmic scale, it is important to realise that a soil with pH 4 is 10 times more acid than a soil with pH 5! Acid soils may be classified as those soils with a pH of less than 7 (i.e. neutral pH). However, this has little value in practice, since most plant species will only show reduced growth at substantially lower pH. Thus, soils with a pH of less than 5.5 may be regarded as acid (i.e. those in which growth of many plant species is adversely affected).

Acid soils occupy about 30% of global land surfaces, and predominate in two major regions of the world: humid temperate forests and humid tropics and subtropics (von Uexküll and Mutert 1995). However, many soils in other areas are acid also. Tropical acid soils alone comprise approximately two billion hectares, or 14%, of the total ice-free area of the world. Approximately 40% of the world’s arable soils (i.e. those on which crops may be grown) are acid.

Eight regions of Australia have been identified in which agricultural production is reduced by acid soils (Figure 16.15). In southern Australia, these include the wheat and sheep belt of Western Australia, the southern portion of South Australia, cropping and grazing lands of southeastern South Australia along with the western, central and northeastern districts of Victoria. There are also about one million hectares of highly acidic soils in Tasmania. Further north in New South Wales, the cropping and grazing regions of the Riverina and southwestern slopes contain many acid soils, as do the tablelands and wet sub-tropical coastal farming regions of New South Wales and Queensland. Soils of the tropical wet coast of Queensland and adjacent intensively cropped and grazed areas are also acid to a considerable extent.

Overall, acid soils are most common where rainfall exceeds 450 mm year^–1 and more than 80 million hectares of the most productive agricultural land in Australia are acidic, with more than 40% of this land being highly acidic (Land and Water Resources Research and Development Corporation 1995). Acid sulphate soils of coastal areas may also acidify when drained for crop production or urban use, with consequent effects on estuarine aquatic life. This most noticeably results in fish kills, often following rains after a dry period during which oxidation of soil S has occurred.

Acid soil infertility in Australia is now recognised as an insidious and invisible form of land degradation. Worldwide, soil nutrient decline and acidification are often the most easily visible forms of land degradation. A landmark Australian study showed that soils under subterranean clover pasture acidify, with a decrease from a soil pH of around 6.0 in virgin soils to pH 5.2 where pasture had been grown for more than 30 years (Table 16.8). Subsequent studies have shown that the acid addition rate over extensive areas of New South Wales ranges from near zero to 3–5 kmol H⁺ ha^–1 year^–1, with up to 20 kmol H⁺ ha^–1 year^–1 in some exploitative systems (Helyar et al. 1990). With an acid addition rate of 4 kmol H⁺ ha^–1 year^–1, there would be a decrease of about 1 pH unit in the surface 30 cm of a sandy loam within 30 years. This would take about 120 years in more strongly buffered clay soils.

16.5.2  Aluminium and manganese toxicities

Soil pH has a marked effect on nutrient availability (Figure 16.2) by affecting both solubility of soil minerals present and the ability of plant roots to absorb nutrients. Generally, a decrease in soil pH increases availability of cations, especially essential micronutrients, Fe, Mn, Cu and Zn. In contrast, availability of Mo decreases, often to such an extent that acid soils are deficient in plant-available Mo. Availability of P also decreases at low pH via fixation with hydrous oxides of Fe and Al, although reactions with Ca can also reduce P availablility around pH 8.5 (Figure 16.2).

(a)  Aluminium toxicity

Al toxicity has been recognised for over 60 years. Shortly after the turn of the twentieth century, soil scientists found Al present in solution leached from soils. However, later attention was given to hydrogen clays, and following development of glass electrodes research emphasis was placed on soil pH for many years — an example of technology driving science! Despite publication of research results between the late 1920s and late 1960s, deleterious effects of Al on plant growth did not attract interest again until the early 1970s. Some of those findings are outlined here.

Al is the most common metal in the earth’s crust (8% of dry mass) and comprises some 7% of soils, where it has a complex chemistry. Al is an important component of aluminosilicate compounds, including clay particles. As soils acidify, Al dissolves from these solid forms, and enters solution with the potential to then become toxic (Table 16.9). Not all Al in the soil solution is toxic, however, providing challenges to biologists to identify those forms that are. Those shown to be toxic to plants include the inorganic Al monomers (Al³⁺, AlOH²⁺, Al(OH)₂⁺) and the inorganic polycation AlO₄Al₁₂ (OH)₂₄ (H₂O)₂₄⁷⁺ (known as Al₁₃).

The concentration of Al in the soil solution is often low (<50 µM), and activities of monomeric Al in the soil solution that reduce root growth have been found to range from 4 to 15 µM. A further challenge to analytical chemists is to develop techniques able to discriminate between toxic and non-toxic forms of Al, made all the more difficult because of the low concentration of toxic Al. This concentration has been calculated to be about 0.1 g Al in 1 m³ of soil (c. 12 µM Al or one-third of one part per million in the soil solution). The same 1 m³ of acid soil would contain a total of 70 kg Al.

Solubility of Al decreases dramatically with an increase in soil pH (Figure 16.16a), and the concentration of inorganic monomeric Al in solution is also affected by organic and inorganic anions. Complexes of Al with organic ligands of low relative molecular mass, especially citric and malic acids, are also considerably less toxic. Tolerant plants benefit from such chelating mechanisms. In addition, organic matter increases the concentration of fulvic and humic acids, resulting in the formation of Al fulvate and Al humate. These complexes are considerably less toxic than the inorganic monomeric species.

Biological effects of ions are often better related to activ-ity in solution rather than to concentration. In addition to soil pH effects on Al solubility, speciation of Al in solution changes with pH (Figure 16.16b). At pH 4.0, most of the inorganic monomeric Al is present as Al³⁺; this decreases with an increase in pH while the activities of the hydroxy Al species (AlOH²⁺, Al(OH)₂⁺) increase. At alkaline pH, aluminate ions (e.g. Al(OH)₄^–, Al(OH)₅^2–) enter into solution but are not toxic to plant roots (Kinraide 1990).

Soluble Al in solution has a rapid effect on root growth, with the effects often visible within 2 d. Microscopically, decreased root growth may be evident within a few hours of exposure to Al, and changes in Golgi apparatus activity have been documented to occur within 5 h of placing roots in a solution containing Al. To be toxic, the root tip must be exposed to Al (Ryan et al. 1993).

While the toxic effect of Al has been known for more than 60 years, the biochemical basis of Al toxicity has not been clarified. However, Al is known to exert its primary toxic effect on roots, initial effects including a reduction in root length (Figure 16.17) (through reduced cell elongation) and damage to cortical cells of the root epidermis near the root tip. Proliferation of root hairs, important for uptake of water, essential nutrients and for infection by N-fixing rhizobia in legumes, is severely reduced by Al at concentrations lower than those that reduce root growth. Proposals for the primary toxic effect have included reactions of Al with components of the cell wall, plasma membranes, cytoplasm and nucleus (Kochian 1995).

Visible effects of soluble Al on plant tops are considered to be a secondary effect through reduced nutrient uptake. Symptoms include those similar to deficiencies of Ca, Mg, Fe, and P, probably as a result of decreased root proliferation and of reduced root activity. Further secondary effects include a reduction in uptake of water, increased sensitivity to drought and decreased N₂ fixation by nodulated legumes.

Plant species differ markedly in tolerance to soluble Al in the soil solution. As with toxicity, the biochemical basis of genetic tolerance of Al toxicity is not clearly understood. However, malic acid excretion by root apices of Al-tolerant wheat is known to be five- to ten-fold greater than that excreted by Al-sensitive wheat. Malic acid presumably complexes soluble Al, making it less toxic (Delhaize et al. 1993), and that capacity for excretion co-segregates with Al tolerance in progeny from crosses between near-isogenic lines. By implication, a single major gene is probably responsible for an Al tolerance that is functionally linked to Al stimulation of malic acid excretion (Delhaize et al. 1993).

(b)  Manganese toxicity

Mn, an essential element for plants (Section 16.3), is about the tenth most abundant element in the earth’s crust. Like Al, Mn chemistry in acid soils is complex, affected by both soil pH and soil redox potential. Mn occurs in rocks mostly in co-ordination with O²⁺ and as the divalent Mn²⁺ in soil solution and in natural waters. Biotic and abiotic oxidation occurs readily so that Mn does not generally occur to excess except in acid soils or in waterlogged soils (assuming there are sufficient Mn-containing minerals). Mn toxicity may occur following soil sterilisation due to loss of microorganisms that normally oxidise Mn²⁺.

Al is a root toxin, whereas high Mn is mainly toxic to shoots (at very high Mn concentration, root growth may be affected directly). Thus, unlike Al, which often has an immediate effect on root proliferation, Mn must accumulate in shoots to become toxic. Three types of symptoms result from Mn accumulation: dark-brown, necrotic spots on lower leaves, distortion of expanding leaves (possibly an induced Ca deficiency) and chlorosis of young leaves. Such chlorosis is often interpreted as an Fe deficiency due to reduced Fe uptake by roots due to excess Mn (Figure 16.8).

Considerable genetic differences in tolerance to high concentrations of plant-available Mn exist among plant species, and even among lines within a species. Such variation may occur via differences in root exclusion of excess Mn, complexation of Mn within roots, or shoot tolerance of high Mn. Sunflower, watermelon and cucumber all show shoot tolerance and even excrete Mn in a biologically inactive form around trichomes (hairs) on leaves and stems (Blamey et al. 1986).

External Mn concentrations at which plant growth is reduced also vary greatly among plant species. In carefully controlled solution culture (Edwards and Asher 1982), the critical external concentration (i.e. the concentration required for 10% reduction in plant dry mass) in the two most sensitive species, maize and wheat, was 1.4 µM Mn (Table 16.10). In contrast, sunflower growth was only reduced with 65 µM Mn in solution. Likewise, the critical internal Mn concentrations varied also, from 200 mg kg^–1 in maize to 5300 mg kg^–1 in sunflower. Although cowpea and bean had a similar external critical concentration, cowpea was able to tolerate a much higher tissue Mn. A higher retention of Mn in roots of soybean enabled this species to tolerate a higher external Mn concentration than cotton.

(c)  Countering adverse effects of acid soils

Acid soil problems have been traditionally corrected via application of agricultural lime (CaCO₃) or dolomite (CaCO₃ + MgCO₃). Such amelioration raises soil pH, reducing Al and Mn concentrations in the soil solution, as well as adding the essential nutrients Ca and Mg. Lime or dolomite is generally required to raise soil pH to a level at which Al or Mn toxic-ities no longer affect plant growth (often above pH 5.5). Rates required vary considerably depending on soil type, with clay soils requiring higher rates to increase soil pH (greater buffering capacity referred to earlier in connection with acidification). Moreover, lime requirements for sustainable agriculture vary with farming systems. For example, lime required (kg CaCO₃ ha^–1 year^–1) to balance net acid accumulation is about: 0.8 for wool, 6.0 for lamb and 7–20 for
cereal enterprises (Slattery et al. 1991). Even though cereal production is not greatly acidifying, Australia’s average wheat crop of 15 million t would have removed alkalinity equivalent to 135000 t CaCO₃ as well as 6000 t Ca, 18 000 t Mg and 66 000 t K (along with 330 000 t N, 42 000 t P and 26 000 t S) from the land. Most of the crop was used for human consumption either in Australian cities or overseas. There would be little return of these nutrients to their original fields.

Application of lime in Australia falls far below that required to balance losses of alkalinity. Annually, c. 0.5 million t of lime is used in Australia, but more like 2.25 million t are needed annually as prophylactic dressings (Land and Water Resources Research and Development Corporation 1995). Overliming is a potential problem, especially on sandy soils, due to reduced availability of essential micronutrients, especially Zn, while root diseases such as ‘Take all’ of cereals are exacerbated.

Amelioration of some acid soils is possible via application of gypsum (CaSO₄.2H₂O) which provides a readily soluble form of Ca. Leaching soluble Ca applied as gypsum may improve root growth in acid subsoil horizons. Application of Mo is also beneficial where this essential micronutrient is in short supply due to low pH.

Finally, breeding and selection for crop tolerance to acid soil limitations has immediate application in agriculture, but an understanding of factors involved is crucial because tolerance to one factor limiting growth on acid soils (Al toxicity) does not imply tolerance to another such as Mn excess or Mo deficiency.

(d)  Concluding remarks

Acid soil infertility is a major limitation to plant growth worldwide, especially in the humid tropics, subtropics and temperate zones. Infertility results primarily from toxicities of Al or Mn and from deficiencies of Ca, Mg, K, P and Mo. While there are many soils that are naturally acid, farming practices and industrial processes contribute to increased soil acidification. These include use of N fertilisers, especially where they exacerbate the loss of basic cations through leaching and crop removal, and growth of legumes, which increase soil N status. Acid rain is important in industrialised regions.

Two complementary approaches may be used to overcome the adverse effects of soil acidity. The first is amelioration of acid soils through application of lime or gypsum to arrest degradation occasioned by a loss of basic cations. Unless these basic cations are replaced, land degradation through acidification and nutrient decline is inevitable. The second is a quest for genotypes better adapted to acid soil conditions. Genetic ap-proaches alone simply buy time and will result in further soil acidification, but do offer a beneficial trade-off: tolerant plants would draw water and nutrients from acid subsoils which are difficult, if not impossible, to ameliorate any other way.

16.5.3  Serpentine soils and mine tailings

Certain rocks, low in silica but rich in ferromagnesium minerals, give rise to soils of typically high pH known generically as ultramafic or, less accurately, serpentine soils (the mineral is mottled green and resembles a snake’s skin). Such soils are rich in Cr, Co, Ni, Fe and Mg, but severely deficient in N, P, K, Ca and possess a high Mg:Ca ratio.

These chemical features select for a highly characteristic flora which often shows stunted growth with chlorotic foliage, and morphologies typical of desert perennials. Such species usually result in clearly defined boundaries between serpentine and adjacent vegetation.

Even more remarkable, serpentine species frequently accumulate prodigious quantities of Cr, Co and Ni, and can even serve as indicator plants for underlying lithology. Serpentine deposits plus associated plant species occur in Tasmania and Queensland, but notable in eastern Australia are the Coolac–Kiandra deposits near Tumut in New South Wales that are dominated by Casuarina stricta, Ricinocarpos bowmanii and Xanthorrhoea australis in place of the Eucalyptus melliodora woodland on adjacent granitic soils. Lyons et al. (1974) provide further details on that deposit, and explain that ‘... X. australis strongly favours soils high in magnesium and low in copper, whereas the distribution of C. stricta appeared to be controlled mainly by the high potassium and nickel values in the soils...’

Within Australia the most extensive deposits are adjacent to the eastern goldfields of Western Australia. Vegetation surveys in Western Australia also show plant associations that reflect lithology. For example, a belt of Eucalyptus aff. cylindrocarpus woodland coincided with an Ni anomaly, while soils high in both Ni and Cr supported Melaleuca laterifolia. Certain plant species thus serve as indicators of soil metal content, and Ni deposits lead to sharp discontinuities in species distribution (see Table 16.11 for more details on soil–plant correlations).

Ernst (1974), cited in Brooks (1987), defined the Hybanthion florbundii Alliance to cover the serpentine vegetation of Western Australia. In such communities, the Alliance commonly replaces eucalypt woodland more common from Kalgoorlie–Kambalda to Leonora, and is characterised by the Ni-hyperaccumulator Hybanthus floribundus and its associated species Grevillea acuaria. H. floribundus was only the third such plant to be documented worldwide, and this species is a reliable indicator of nickeliferous soils for two reasons. First, H. floribundus is restricted to ultramafic outcrops and their drainage areas. Moreover, within these shrub communities, a significant positive correlation exists between stand density and soil Ni content, especially on soils where Ni content exceeds 880 ppm. Second, all subspecies and forms of H. floribundus hyperaccumulate Ni, and in one case Co, even when soil levels are low (Table 16.12).

Despite such unusual nutritional physiology where Ni can accumulate to over 5000 ppm dry mass, no selective advantage for plant growth and reproduction from Ni accumulation is obvious, although herbivores might be discouraged by high levels of tissue Ni (see Batianoff et al. 1990 for more details). Mechanisms that permit plant tolerance to such hyperaccumulation have yet to be defined.

Serpentine soils occur naturally, but other metal-containing soils have been formed as a consequence of mining and smelting of ores (e.g. Cu, Pb and Ni), or following coal mining where a highly acidic overburden (pH <4.5) predisposes to Al and Mn toxicity (Section 16.5.2). Plant growth will then be limited by both heavy-metal toxicity and effects of Al or Mn, as well as by deficiency of essential nutrients such as N, P, K, Cu and Mg. Such deficiencies can of course be alleviated by fertiliser applications, which also carry a potential side benefit of heavy-metal attenuation via formation of complexes (e.g. Al–PO₄).

Prospects for successful mine site rehabilitation are thus improved on both counts, but increasingly successful colonisers have been shown to have mycorrhizal associations (Khan 1978; Gardner and Malajczuk 1988). Mycorrhizal fungi are widely recognised as enhancing host plant uptake of sparse nutrients (Section 16.4.1), but they can also detoxify heavy metals, and strong selection for tolerance to Ni, Al, Zn and Cu has been reported from Europe and North America (see Jones and Hutchinson 1988). Similarly, selection of metal-tolerant isolates (ecotypes) in the ectomycorrhizal fungus Pisolithus tinctorius has been noted for Al-containing soils in Western Australia, where Al tolerance of isolates was positively correlated with Al concentration in source soil (Egerton-Warburton et al. 1993).

Root anatomy of Eucalyptus rudis seedlings following inoculation with P. tinctorius reveals a well-developed mycorrhizal sheath, a larger root diameter and intact cells compared to smaller root dimensions and necrotic tissue in non-mycorrhizal roots.

 

Benefits of mycorrhizal associations in metal-containing soils include improved survival of seedlings, greater growth and higher nutrient content (Table 16.13). One notable difference between inoculated and non-inoculated eucalypt seedlings concerns internal distribution of Al rather than total content per plant. The mycorrhizal sheath of inoculated plants accumulates Al, resulting in lower concentrations in roots and leaves. By contrast, non-mycorrhizal plants show strong accumulation in all tissues (Figure 16.18).

In addition, the higher P content of mycorrhizal seedlings (Table 16.13) also comes from accumulation and storage as polyphosphate granules by the mycorrhizal sheath (see Ashford et al. (1986) and Orlovich et al. (1989) for more information on these structures).

Polyphosphate granules form an effective sink for Al in E. rudis–P. tinctorius associations, and conceivably formation of Al–PO₄ complexes in these granules represents a beneficial detoxification site for excess Al in these low pH soils.

K content in mycorrhizal plants was also much higher (Table 16.13), and was associated with a minor reservoir of K in root sheaths, and a major localisation in leaf mesophyll. Xylem-mobile elements such as K would be expected to accumulate at vein endings in this way (Section 5.2).

Mycorrhizal associations have greatest impact on plant Ca and Mg content and distribution (Table 16.13, Mg in Figure 16.19), and such significant increases might affect nutritional physiology in several ways. They could (1) facilitate uptake of Ca and Mg from acidic soils frequently impoverished with respect to Ca and Mg; (2) offset adverse effects of Al on uptake of Ca and Mg by roots; (3) ameliorate Al toxicity through enhanced Ca uptake.

In effect, efficient acquisition and distribution of Ca might be a prerequisite for Al tolerance. Consequently, any selection pressure experienced by mycorrhizal fungi in high-Al soils which has resulted in improved tolerance to heavy metals probably operates via the capacity of mycorrhizal fungi to enhance Ca and Mg uptake into their host plants.