Micronutrient Micnobit and Salt Fertilization on Lettuce
The impact of micnobit (nano- and micro-size particle) and salt fertilizer application on soil and leaves was tested on growth, nutrient content, and metabolites of lettuce under greenhouse conditions. No distinct differences were observed between the treatments, whether fertilizer type or mode of application. Nutrients were found to relocate from root to shoot and vice versa, and the presence of micnobits of some elements was detected in the plants. Vitamin C content was not affected, and over more than 60, yet to be identified, metabolites revealed a two-fold difference.
Effect of micronutrients on cucumber postharvest quality
Micronutrients are required in small amounts for the proper functioning of plants, due to their involvement in numerous metabolic processes. They are known to enhance growth, raise nutritional value of produce, enhance resistance to diseases and tolerance to drought. Michail Kendristakis recently did his MSc thesis at the Horticulture and Product Physiology group of Wageningen University, the Netherlands, on the effects of supplementing micronutrients to cucumber fruit on fruit quality characteristics and shelf-life. In his experiments, calcium and boron were applied twice to the fruit in the weeks before being harvested.
Harvested fruit from both the calcium and boron treatments were significantly greener and firmer than the water-treated control fruits. In addition, treated fruit had slightly higher sugar content. During the postharvest storage, micronutrient-treated fruit remained firmer and showed decreased water loss leading to an extension of the shelf life compared to the control fruit.
Calcium is an essential element for the cell wall structure and for the interconnection between the cellulose fibrils. Calcium may also improve membrane integrity. Boron stimulates the synthesis of hemicelluloses and other related cell wall materials. This explains the positive effect of calcium and boron on fruit firmness. In addition, treated fruit had a higher percentage of closed stomata which may have restricted the postharvest water loss and resulted in fruit that stayed firm and fresh for longer.
These finding are important in strategies to reduce food waste that currently amount to one third of all edible food.
MSc thesis. Michail Kendristakis. The effect of micronutrients on cucumber postharvest quality. Wageningen University, 2017.
Supervisors: Ernst Woltering, Nikolaos Ntagkas and Prem Bindraban
Micronutrients Mitigate Drought Stress
Composite micronutrient nanoparticles and salts decrease drought stress in soybean
Micronutrients provide multiple benefits in crops, some, being involved in plant-soil-water relations. Our recent paper demonstrates the effects of micronutrients in mitigating drought stress in soybean. A micronutrient formulation of zinc (Zn), copper (Cu) and boron (B) was delivered as nanoparticles or as ions from salts, after which drought was imposed on the plants over several weeks. Drought severely reduced soybean growth, grain yield and uptake of several key nutrients, including nitrogen (N), potassium, Zn and B. However, treatment of the plants with micronutrients under drought mitigated these reductions. Nitrogen uptake was significantly increased, which provides a potential strategy to increase plant N use efficiency using micronutrients, and reducing N fertilizer losses to water and air. Similarly, Zn uptake and grain Zn content were significantly enhanced under drought by the formulations. Fortification of Zn in food crops by agronomic practice may therefore be an effective strategy for increasing the nutritional quality of edible produce under water limiting conditions. The full article can be found here.
Micronutrients Stakeholder Workshop
VFRC co-commissioned report
Micronutrients for improving harvests, human nutrition, and the environment
This essay considers the potential of agronomic biofortification by exploring the effectiveness of micronutrient fertilization to improve crop productivity, the nutritional quality of edible plant parts, the bioavailability for human uptake and human nutrition. Pathways to make these fertilizers available to resource poor farmers in sub-Saharan Africa are briefly explored. This paper is a background document for the participants of the Micronutrients Stakeholder Workshop on April 5 2016, organized by the Food & Business Knowledge Platform in collaboration with IFDC (VFRC) and Wageningen UR in The Netherlands.
Anne W. de Valença and Anita Bake, 2016. Micronutrient management for improving harvests, human nutrition, and the environment. Background document for stakeholder workshop on micronutrients (05 April 2016). Wageningen University and Research.
Fortification of Micronutrients for Efficient Agronomic Production
A recent VFRC review article shows that micronutrients (copper, iron, manganese, molybdenum, nickel, zinc, boron and chlorine) can be used as a one-stop shop for enhancing crop nutritional quality, yield, biomass production and resiliency to drought, pest and diseases. These positive effects can be observed with or without NPK fertilization. The article discusses the uptake by plants of micronutrients as tiny (nano and micro-size) particulate materials, coined “micnobits,” relative to their conventional uptake in ionic forms. Packaging of micronutrients as micnobits was shown to have profound effects on crop responses and fertilizer use efficiency compared to conventional salts or bulk oxides, and to create alternative nutrient delivery strategies to plants such as uptake through above ground parts.
Citation: Dimkpa, C. and P.S. Bindraban, 2016. Micronutrients fortification for efficient agronomic production. Agronomy for Sustainable Development. In press. DOI 10.1007/s13593-015-0346-6
How Selenium Helps to Improve Human Health
The global importance of selenium (Se) deficiency to human and animal health has been recognized for decades, but strategic Se fertilization interventions were lacking. Our meta-analysis approach based on 243 experiments performed during 1960 to 2014 confirms the high impact of Se-fertilization as an effective agronomic bio-fortification strategy. Fertilizer formulation, dose and timing affect crop Se uptake, with highest uptake efficiencies for foliar and selenate based fertilizers. Minor influence of soil organic matter, total soil Se levels and acidity suggests that other agroecosystem properties like climate and bioavailable Se should be considered to optimize Se-fertilization. Tailor-made strategies are essential to combat the current low recoveries and the scarce resource availability.
Citation: Ros, G. H., A. M. D. van Rotterdam, D. W. Bussink & P. S. Bindraban, 2016. Selenium fertilization strategies for bio-fortification of food: an agro-ecosystem approach. Plant Soil. DOI 10.1007/s11104-016-2830-4.
VFRC Report 2015/1: Beyond N and P: Toward a Land Resource Ecology Perspective and Impactful Fertilizer Interventions in Sub-Saharan Africa
Food for thought about complexity for nutrient blending
Antagonistic and synergistic effects between nutrients in soil availability and uptake by plants can dramatically affect yield. This report presents experimental results with multiple nutrients and construes initial indications for the existence of complex nutrient relations under field conditions. Whereas the scientific foundation is yet to be firmly established, it suggests that, rather than absolute levels of nutrients present in the soil, it is their proportion expressed as ratios that impacts crop yield. These ratios are particularly important among the basic cations (Ca, Mg and K), between P and micronutrients and among the micronutrients themselves. Such ratios are shown to govern the ecological diversity of vegetation and spatial pattern of soils. It is therefore essential to include all essential nutrients in agronomic and fertilizer research. Limited amount of optimal ratios of (micro)nutrients tuned to local soil chemical properties can have large yield impacts and result in higher fertilizer uptake efficiency.
Citation: R. Voortman and P.S. Bindraban, 2015. Beyond N and P: Toward a Land Resource Ecology Perspective and Impactful Fertilizer Interventions in Sub-Saharan Africa. VFRC Report 2015/1. Virtual Fertilizer Research Center, Washington, D.C. 49 pp.; 15 tables; 17 figs.; 2 text boxes; 67 ref.
VFRC Report 2015/2: Nanoscale Micronutrients Suppress Disease
Soil-borne pathogens cause major crop losses in global agriculture, adding up to billions of dollars of lost productivity each year. Alternative strategies applying nanotechnology in agriculture have focused on smart delivery systems and nanosensors to increase efficiency. However, low micronutrient availability in the soil and poor in planta translocation can limit the utility of amendment strategies. The current report addresses a potential strategy that has not been adequately explored: the use of nanoscale amendments (metal, metal oxide, carbon) to suppress crop disease and subsequently enhance growth and yield. The current regulatory perspective for such applications is also discussed.
A peer reviewed article based on this report is available here.
Citation: Servin, A., W. Elmer, A. Mukherjee, R. De La Torre-Roche, H. Hamdi, J.C. White and C. Dimkpa
Nanoscale Micronutrients Suppress Disease, 2015. VFRC Report 2015/2. Virtual Fertilizer Research Center, Washington, D.C. 33 pp.; 4 tables; 1 figs.; 118 ref.
VFRC Report 2015/3: The Application of Nanotechnology for Micronutrients in Soil-Plant Systems
Billions of people and many soils across the planet suffer from MN deficiencies, impairing human health. The micronutrients (MNs) use efficiency (MUE) by crops of fertilizers is < 5% due to a lack of synchronization between the fertilizer-MNs release and their crop demand during growth.
Nanotechnology may transform fertilizers. Nanomaterials (NMs), when used as nutrients, are taken up and mobilized by plants, although their effects on crops, remain unknown. A novel model for a MN Intelligent NanoFertilizer (INF) is presented that may enhance MUE and food quality by enabling the synchronization of MN release from fertilizers according to crop demand.
Citation: C.M. Monreal, M. DeRosa, S.C. Mallubhotla, P.S. Bindraban and C. Dimkpa, 2015.
The Application of Nanotechnology for Micronutrients in Soil-Plant Systems. VFRC Report 2015/3. Virtual Fertilizer Research Center, Washington, D.C. 44 pp.; 1 tables; 5 figs.; 270 ref
VFRC Report 2015/4: Plant Exudates for Nutrient Uptake
Many plant strategies for acquiring nutrients rely on the secretion of root exudates. In this report, important classes of exudates related to nutrient acquisition are identified, their mechanisms of enhancing nutrient availability are explained, and their effectiveness in making nutrients in the soil more available to plants are discussed. A meta-analysis of literature data on plant exudation responses to nutrient deficiencies has been included. Type of exudate and increase in exudation rate under nutrient deficiency strongly depend on the species or even cultivar, and on the deficient nutrient. Possibilities for further utilizing the potential of root exudates for nutrient acquisition in agricultural systems should be explored.
Citation: D.H. Keuskamp, R. Kimber, P. Bindraban, C. Dimkpa and W.D.C. Schenkeveld, 2015. Plant Exudates for Nutrient Uptake. VFRC Report 2015/4. Virtual Fertilizer Research Center, Washington, D.C. 53 pp.; 10 tables; 18 figs.; 179 ref.
VFRC Report 2015/5: Effects of Nutrient Antagonism and Synergism of Fertilizer Use
The role of mineral nutrients in crops is influenced by antagonistic or synergistic interactions among nutrients. In this report, scientific articles quantifying synergistic, antagonistic, additive, non-significant or negative responses in different plants were identified. The study indicated some general trends: (a) when two nutrients are deficient, a large increase in yield can be expected by diminishing the deficiency; (b) for most macronutrients the mutual interactions on yield levels are synergistic; and (c) antagonistic effects on yield are often found between divalent cations. Because of the limited number of crops, nutrients, soil types and climates in which these interactions have been studied, care should be taken in extrapolating individual results to other situations.
Citation: R.P.J.J. Rietra, M. Heinen, C. Dimkpa and P.S. Bindraban, 2015. Effects of nutrient antagonism and synergism on fertilizer use efficiency. VFRC Report 2015/5. Virtual Fertilizer Research Center, Washington, D.C. 42 pp.; 16 tables; 1 fig.; 229 ref.
The Need for a Paradigm Shift in Fertilizers
The discovery and use of fertilizers has been one of the key drivers for increasing crop yield, agricultural productivity and food security, yet it comes at a high environmental and economic cost. Applying existing fertilizers, primarily NPK, under the right conditions can lessen these issues, but overall progress is inadequate for the desired transformation toward sustainable, nutritive, resilient and climate-smart agriculture.
This paper reflects on current fertilizer use and discusses the need and possibilities for a paradigm shift in fertilizer research and development. A number of innovative changes in the way we apply fertilizer have the potential to resolve problems such as mineral loss and affordability to poor farmers. However, a current lack of focus in fertilizer research and development hinders the progress of these new techniques.
The authors of this report propose a concerted research and development effort among research institutions and private sector players, orchestrated by public debate and policy to further the advancement of these game-changing fertilizers.
Citation: Prem S. Bindraban, Christian Dimkpa, Latha Nagarajan, Amit Roy, Rudy Rabbinge, 2015. Revisiting Fertilisers and Fertilisation Strategies for Improved Nutrient Uptake by Plants. Biology and Fertility of Soils 51 (5): 897-911.
p style=”text-align: center; font-size: 25px;”>VFRC Generates High-Resolution Soil Micronutrient Maps
Based on VFRC review studies and on on-farm trials conducted by IFDC, the VFRC has prioritized R&D on micronutrients. Yet, the lack of awareness about soil fertility constraints is a major limitation to developing sound liming and fertilizer recommendations in sub-Saharan Africa, certainly so for micronutrients. VFRC has, therefore, engaged in research in collaboration with ISRIC–World Soil Information, IFDC and local organizations in Rwanda and Burundi to develop detailed soil maps produced with the most recent geo-spatial statistical methodologies. Maps of primary (P, K), secondary (Ca, Mg, S) and micronutrients (Cu, Zn, B), as well as pH, soil acidity (Al+H), effective CEC and organic matter were generated for the 0-20 cm soil layer by means of digital soil mapping using random forest models at a 250 m spatial resolution for Burundi and Rwanda. The maps form the basis to support soil fertilization strategies, i.e., where to apply which combination of nutrients.
Citation: María Ruipérez González, Bas Kempen, Prem S. Bindraban, Sandra Wolters, Cyrille Hicintuka, Marcien Nibasumba, Zacharie Nzohabonayo, John Wendt, Oscar Nduwimana, John Veerkamp, 2015. Digital mapping of soil nutrients for the Republics of Burundi and Rwanda. Poster presented at the Wageningen Soil Conference, 23-27 August 2015, Wageningen, The Netherlands.
VFRC Report 2015/6: Preliminary Evaluation of the Feasibility of Using Geospatial Information to Refine Soil Fertility Recommendations
IFDC carries out fertilizer field trials at hundreds of georeferenced locations throughout eastern and southern African countries. This report uses geo-spatial methodologies to make soil maps and to analyse for spatial pattern of yield responses along with the factors causing the differences in the responses.
These insights can support location- or region-specific fertilizer targeting as an alternative to blanket recommendations. The information may also be valuable for fertilizer companies and other actors in the chain at regional scale in finding where to sell which fertilizer composition.
Citation: Kempen, B., Vereijken, P.F.G., Keizer, L.P.C., Ruipérez González, M., Bindraban, P., Wendt, J., 2015. VFRC preliminary evaluation of the feasibility of using geospatial information to refine soil fertility recommendations. VFRC Report 2015/6. Virtual Fertilizer Research Center, Washington, D.C. 67 pp.; 20 tables; 31 fig.; 30 ref.; 4 appendices.
VFRC Report 2014/1: Beneficial Organisms for Nutrient Uptake
This review about beneficial soil organisms for plant nutrient acquisition reveals the fascinating beauty of soil biology and the amazingly complex interactions among plants, soils, micro-organisms and nutrients. Generally speaking, micro-organisms may help plants to better scavenge several nutrients from the soil and reduce hazards such as drought or toxicity against heavy metals. Some micro-organisms can keep away harmful organisms, improving plant fitness. Some microorganisms are even considered to be biofertilizers. Therefore, maintaining a diverse population of micro-organisms by adequate management (including prevention of overuse of mineral fertilizer) may be beneficial in the long run.
The strong and multiple interactions imply, however, that human interventions targeting the exploitation of specific beneficial aspects would be highly specific regarding plant species, soil, micro-organism and nutrients. The beneficial impact of currently available inoculants seems to vary greatly due to these complex interactions, and various commercially available products may lack rigorous scientific evidence explaining their impact. Yet some generic finding may give leads for interventions for increased nutrient uptake via microorganisms. For instance, with specific nitrogen-fixing bacteria, evidence is overwhelming that interactions between plants and micro-organisms are beneficial for nutrient supply to plants. Furthermore, partial application of nutrients through leaves may help to reduce the negative impact of soil fertilization on symbiosis between plants and beneficial micro-organisms. Hence, understanding the world of soil microbiology and exploiting it to the benefit of more sustainable production remains worth investigating.
Citation: Nina Koele, Thomas W. Kuyper and Prem S. Bindraban, 2014.Beneficial organisms for nutrient uptake. VFRC Report 2014/1, Virtual Fertilizer Research Center, Washington, D.C. 63 pp.; 4 figs.; 330 ref.
VFRC Report 2014/2: Eliminating Zinc Deficiencies in Rice-Based Systems
Zinc deficiency in crop production and in human populations is a wide-spread and serious problem, especially in rice-based systems. This reports presents an overview of the soil factors controlling plant-available Zn, explains the synergistic or antagonistic behavior among nutrients on their uptake by plants and discusses the mobility of Zn within a plant. This holistic, comprehensive view of the behavior of Zn from the fertilizer through to the plant and human intake provides leads as to how Zn nutrients could be best supplied to rice plants. The report also evaluates biofortification of rice against other strategies to resolve Zn deficiency in human consumers depending on rice as staple food.
Citation: A. Duffner, E. Hoffland, T.J. Stomph, A. Melse-Boonstra and P. S. Bindraban, 2014.
Eliminating Zinc Deficiency in Rice-Based Systems. VFRC Report 2014/2. Virtual Fertilizer Research Center, Washington, D.C. 35 pp.; 1 table; 5 figs.; 1 text box; 200 ref.
VFRC Report 2014/3: Se Fertilization: An Agro-Ecosystem Approach
Selenium (Se) is an essential micro-nutrient with 0.5 to 1 billion people experience major health problem due to deficient intake. This research identifies factors for developing a decision support tool for determining when application of Se fertilizer would be effective. The meta-analysis done in this research reveals that fertilizer strategies are key to increase Se uptake of staple food crops. Adapting fertilizer strategies to site specific agro-ecosystem properties might increase the uptake efficiency from 10% up to 50%. Most relevant soil properties controlling Se uptake include soil acidity and redox potential. Agronomic practices such as liming, irrigation and basic fertilization (nitrogen, phosphorus and sulphur) additionally affect crop Se levels. The chemical form and Se dose are primary drivers of Se uptake whereas foliar applications appears more resource efficient over soil application. Bio-fortifying food items in itself is also an effective avenue for human health improvement but may not reach the neediest.
Citation: G.H. Ros, A.M.D. van Rotterdam, G.D. Doppenberg, D.W. Bussink and P.S. Bindraban, 2014. Se Fertilization: An Agro-Ecosystem Approach. VFRC Report 2014/3. Virtual Fertilizer Research Center, Washington, D.C. 62 pp.; 1 table; 21 figs.; 282 ref.
VFRC Report 2014/4: Establishing a Viable Fertilizer Quality Detection System
Fertilizer adulteration can be a malpractice or unconscious result in the multiple blending steps in the fertilizer chain, ultimately reducing farm livelihood, causing mistrust in the use of fertilizers by farmers and even lead to soil and crop damage and pollution. This research has aimed to develop a quick and cheap method to determine fertilizer nutrient content and trace contamination. Promising results were found with chromatogram imaging, but the method needs further development to be sufficiently accurate, speedy and affordable, revealing the formidable challenge to develop quick and robust methods.
Citation: Perumal, K., Ananthi, S., Arunkumar, J., Sambanda Moorthy, T.A., Karthik, B., U. Singh, and P.S. Bindraban, 2013. Establishing a Viable Fertilizer Quality Detection System, VFRC Report 2014/4. Virtual Fertilizer Research Center, Washington, D.C. 23 pp.; 9 tables; 3 figs.; 8 ref.
VFRC Report 2014/5: Environmentally Friendly Phosphate Fertilizers
Phosphate-solubilizing (PS) Gram-negative bacteria such as Pseudomonads have routinely demonstrated the ability to drive the dissolution of agronomically relevant levels of phosphate rock, thereby promoting plant growth in different studies. This makes such microbes candidates for the industrial development of phosphate fertilizers for crop nutrition. Virtually all P fertilizers are still manufactured via sulfuric acid-based wet processes. This report provides a way forward for the development of phosphate fertilizers leveraging the previously mentioned abilities of PS bacteria. The report calls for an industrial-scale biotechnological intervention to process rock phosphate ores into soluble P fertilizer products, with concrete R&D activities and timelines to arrive at a PS bacteria-enabled crop nutrition product. In view of its biological basis, the use of microbes to dissolve rock phosphate is both an economically and environmentally sustainable technology with increased crop performance benefits.
Citation: Goldstein, A., 2014. Strategies for the Development of Environmentally Friendly Phosphate Fertilizers Based on Gram-Negative Phosphate Solubilizing Bacteria. VFRC Report 2014/5. Virtual Fertilizer Research Center, Washington, D.C. 51 pp.; 5 tables; 27 figs.; 45 refs.
VFRC Report 2013/1: Following the Path of Nutrients in the Leaves
Plant roots absorb nutrients from the soil and transport them to the leaf, where they are assimilated by biological-chemical processes into proteins, fats, vitamins, hormones, etc., for plant growth. Soil is an extremely complex medium, and much of the fertilizer nutrients applied for plant uptake are actually not taken up and are consequently lost to the environment.
Interestingly, leaves can absorb nutrients also. This direct path of nutrients entering the plant near the assimilation point might enhance fertilizer efficiency. However, the efficacy of foliar fertilizer application is ambiguous because little is known about the plant processes governing uptake and utilization.
This paper describes paths of foliar nutrient absorption and outlines possible biochemical pathways of phosphorus (P) and iron (Fe) penetration, absorption and their translocation into various cell organelles where they engage in biochemical processes. This will help in identifying effective fertilizer nutrient composition and in developing next-generation foliar fertilizers.
Citation: Renu Pandey, Vengavasi Krishnapriya and Prem S. Bindraban. 2013. Biochemical nutrient pathways in plants applied as foliar spray: Phosphorus and iron. Washington, VFRC, VFRC Report 2013/1. 23 pp.; 6 figs.; 63 ref.
VFRC Report 2013/2: Fertilizer Uptake Through Leaves – Yet to be Disentangled
Nutrients are taken up by plant roots, but interestingly, leaves can absorb nutrients also. Research is not clear about the effectiveness of foliar fertilizers in agricultural crops and this initial study systematically analyzes the information to improve understanding about the matter.
Foliar fertilizers are applied for a variety of reasons, with greatly differing application methods and uptake efficiencies. Even after leaf penetration the relevance to plant growth and development remains ambiguous, e.g., because some nutrients are not remobilized to other organs once bound to permanent structures where deposited.
The interactions between physical, chemical, environmental and crop-physiological processes, along with metabolic processes, must be understood to arrive at effective foliar interventions.
Citation: Wim Voogt, Chris Blok, Barbara Eveleens, Leo Marcelis and Prem S. Bindraban. 2013. Foliar fertilizer application – Preliminary review. Washington, VFRC, VFRC Report 2013/2. 43 pp.; 9 tables; 2 figs.; 98 ref.
VFRC Report 2013/3: Methodology to Assess the Impact of Fertilizer Strategies on Planetary Boundaries
Fertilizers affect plant growth fundamentally and are essential to feed the world population. Yet, fertilizer use also contributes to eutrophication and greenhouse gas emissions. Overuse will aggravate these side effects, but underuse leads to agro-ecosystem’s degradation, poverty and hunger.
The use of nitrogen (N) and phosphorus (P) fertilizers has been identified as one of the driving forces that push the Earth from its stable geological era of the Holocene into the Anthropocene with unknown implications for life on Earth. Curtailing nutrient losses will therefore have far reaching implications on the Earth’s ecosystem functioning and human health and well-being.
This report describes a methodology to quantitatively link global N and P cycles to four other drivers of global change, being land-system change, freshwater use, climate change and stratospheric ozone depletion. This will allow the assessment of the impact of fertilizer interventions on these drivers, revealing synergies and trade-offs with respect to food security and the environment.
Citation: J.G. Conijn, F.J. de Ruijter, J.J. Schröder and Prem S. Bindraban. 2013.Methodology to assess the impact of fertilizer strategies on planetary boundaries. VFRC Report 2013/3. Virtual Fertilizer Research Center, Washington, D.C. 21 pp.; 1 table; 3 figs.; 42 ref.
VFRC Report 2013/4: Early Growth, a Vital Stage for P Uptake
Soils, either poor or fertile, contain much more P than is required for the yearly uptake. Yet, soil chemical processes fix most P, which leads to very low P concentrations in the soil solution. Crops can scavenge these low concentrations by exploring the soil profile with roots. The total length of the root system is then crucial as it is the constraining factor for P uptake, especially in the early crop stages. This report explores whether the uptake of P can be enhanced during this early stage, thereby stimulating the use of soil P reserves afterwards. Relevant literature is reviewed and reflected on possible measures like seed coating, P placement, foliar P application and the role of exudates to enhance early uptake.
Citation: A.L. Smit, M. Blom-Zandstra, and A. van der Werf and P.S. Bindraban, 2013. Enhancing early root growth to exploit indigenous soil P and fertilizer P. VFRC Report 2013/4. Virtual Fertilizer Research Center, Washington, D.C. 36 pp.; 4 tables; 6 figs.; 98 ref.