Introduction

The study of plant responses to stress has been a central feature of environmental physiologists' attempts to understand how plants function in their natural environment and in particular to explain patterns of plant distribution and their performance along environmental gradients (Osmond et al., 1987). The best of these studies have had a vertical integration from cellular to ecosystem processes (e.g. Björkman, 1981). The primary object of this volume is to bring together contributions from ecologists, physiologists, molecular biologists and plant breeders, who each have their own perspectives on stressful environments and how plants perform in them, and to consider how their understanding of plant responses may be applied by plant breeders. Unfortunately in a book of this size it is not possible to cover all types of stress. In particular, detailed treatment of plant responses to temperature extremes has been omitted as it is the subject of a recent SEB symposium volume (Long & Woodward, 1988), and the extensive potentially relevant work on, for example, pollution, pests and pathogens, and mineral nutrition has been omitted.

In recent years the extended controversy concerning the appropriate terminology to use in studies of plant responses to stressful environments (e.g. Kramer, 1980; Levitt, 1980; Harper, 1982) has often detracted attention from the identification and understanding of underlying principles. Despite this it is useful at this stage to outline the main concepts involved and attempt to provide a generally acceptable common framework for further discussions.

Introduction

On a global basis, drought limits plant growth and crop productivity more than any other single environmental factor (Boyer, 1982). Even in Britain, rain-free periods are frequent enough for irrigation to lead to yield advantages for many agricultural and horticultural crops. Water supply is restricted in many parts of the world and productivity in these environments can only be increased by the development of crops that are well adapted to dry conditions. It is clear that the potential for biotechnological improvement of crop performance cannot be realised until we have identified genes and gene products which are responsible for the desired characteristics of drought tolerance. This in turn cannot occur without a thorough understanding of the biophysical, biochemical and physiological perturbations that are induced by a restricted water supply.

Although plant growth rates are generally reduced when soil water supply is limited, shoot growth is often more inhibited than root growth and in some cases the absolute root biomass of plants in drying soil may increase relative to that of well-watered controls (Sharp & Davies, 1979; Malik, Dhankar & Turner, 1979). It is also commonly observed (e.g. Sharp & Davies, 1985) that the roots of unwatered plants grow deeper into the soil than roots of plants that are watered regularly. Clearly, increases in the density and depth of rooting can help sustain a high rate of water extraction in drying soil (Sharp & Davies, 1985) and may promote substantial improvement in yield in dry years (Jordan, Dugas & Shouse, 1983).

Introduction

Research on genetic manipulation of plants has accelerated dramatically during the past five years. This has exploited ‘natural’ gene delivery systems, leading to the development of vectors, and a number of ‘vectorless’ DNA delivery systems. Further, since plants show extensive powers of regeneration it is, for many species, possible to grow whole plants from genetically engineered cells. Formation of flowers on the genetically engineered plants, followed by pollination, fertilisation, embryogenesis and seed set will then lead to the transmission of the acquired gene to the next generation, provided of course that the gene in question is stably incorporated into the host plant's genome.

In this chapter we first discuss the development of systems for delivering DNA to plant cells. The remainder of the chapter then outlines the ways in which molecular genetics can be applied to research on stress tolerance, giving examples where these techniques are being used.

Systems for gene transfer

For a detailed discussion of DNA delivery systems, readers are referred to the recent very comprehensive review by Walden (1988). Here we concentrate on the two methods most used in stress tolerance studies.

Agrobacterium tumefaciens and its tumour-inducing plasmid

Agrobacterium tumefaciens is a soil-dwelling bacterium which readily infects a wide range of dicotyledonous plants, usually gaining entry via a wound site. Infection causes the growth of a tumour, usually at the original infection site (Fig. 1) and once the tumour is established it will grow in the absence of bacterial cells.

Introduction

Flowers are ephemeral structures and do not themselves suffer from many diseases, though they are points of entry for pathogens. Fruits and seeds on the other hand are a major world food which may be on the plant for a long time and may then be stored. They are subject to a large number of diseases, both during growth and post harvest and this, together with insect attacks, represents an enormous loss (about one-third of production, even with the use of pesticides) which is serious in both economic terms and in the human and animal suffering caused by starvation. If even post harvest losses could be reduced or eliminated the world's food problems would be solved. We can, and do, produce enough food but without expensive storage facilities much is lost, especially in the tropics with ideal conditions for decay but often the inability to pay for chilled stores and other expensive means of storage.

There are a large number of chemicals potentially capable of controlling the spoilage organisms, which are mostly fungi, but there are serious toxicity problems. Fruits are grown to be eaten and, especially with post harvest rots, the problem may be at its most serious close to the point of consumption. There are, therefore, limitations on the sort of chemicals that can be used. There is less of a problem in the field because of the longer time between application and consumption. The same care needs to be taken with biological control agents because application of large numbers of fungal spores or bacteria to protect fruits could lead to the ingestion of their metabolic products or the organisms themselves.

Introduction

The initial section of this chapter will briefly review three attempts to devise predictions of plant responses to stress. The first, Liebig's Law of the Minimum, originates from agricultural research and places strong emphasis upon the identity of the stress most severely limiting plant growth in each environment. The second derives from population biology and focuses upon the ways in which plants under stress differ from each other in demographic responses. The third approach (Plant Strategy Theory) attempts to bridge the gap between ecophysiology and population biology and recognises recurrent forms of ecological and evolutionary specialisation which are frequently associated with characteristic stress responses.

After describing some of the main implications of Plant Strategy Theory for the study of stress responses, brief accounts are provided of two additional dimensions of variation in plant response to stress; these consist of ‘stored growth’ and resistance to mechanical stress.

The search for a predictive model of plant responses to stress

Liebig's Law of the Minimum and its successors

The foundations of intensive modern agriculture and particularly the manipulation of soil nutrient supply owe much to the recognition by Liebig in 1840 that plant yield could be stimulated by recognising the identity of the resource most limiting upon dry-matter production at a given place and time. Since many plant types (e.g. hydrophytes, shade plants, legumes, calcicoles, calcifuges, metallophytes, halophytes) differ in resource requirements according to their evolutionary history, the principle was rapidly accepted in agriculture and in ecology that plant distributions over space and in time are often influenced by the differential responses of plants to variation in limiting resources.

Introduction

Water is, of course, essential for plant growth, but one of the themes of this chapter is that it may not be necessary for plant survival. Although most agronomically important plants are very sensitive to internal water deficits, the majority of plants at some stage of their life cycle are tolerant of desiccation. Few of these have vegetative parts which are desiccation tolerant, but the survival of even so-called drought-evading species, such as the ephemeral desert annuals, rests on the tolerance of their seeds to desiccation.

Desiccation tolerant species may exhibit little or no metabolic activity depending upon the extent of dehydration. In this anhydrobiotic or ametabolic state we are concerned not with metabolic perturbation but with the stability of organelles, membranes and macromolecules in a dehydrated state. However, in the initial period of rehydration, the passage to a metabolically active state poses particular problems if metabolic ‘mayhem’ is to be avoided.

Knowledge of how organisms avoid irreversible damage from dehydration is obviously of importance in the preservation of seeds and germplasm. It may also further our understanding of the role played by water in maintaining structural and functional integrity of membranes and macromolecules. The controlled metabolic shutdown which occurs as organisms pass into a state of anhydrobiosis may provide valuable insights into the functional significance of the metabolic responses to water deficits in mesophytes.

Desiccation tolerant plants

There are representatives of desiccation tolerant species amongst all of the major plant divisions. The water content of many bacterial and fungal spores is low (<25%) and they exhibit great tolerance of desiccation (see Ross & Billing, 1957; Bradbury et al, 1981).

Introduction

Salinity and drought, two environmental stresses frequently found together, are major barriers to productivity of agricultural crops throughout the world. Crops exposed to these stressful environments are observed initially to have reduced growth rates. If the stress is more severe the response is manifested visually in a number of specific and recognisable symptoms, many of which are common to both salinity and drought. However, specific ion toxicity responses (e.g. marginal leaf burn) are observed in plants exposed to excess salinity. Since salinity and drought have many common responses some of the information presented in this review will be combined under the general umbrella of environmental stress.

This chapter discusses the use of tissue and protoplast culture as a means to understand better the cellular processes related to stress tolerance with the expectation that these techniques will provide alternative methods for screening germplasm and assist in identifying useful material for incorporation into crop improvement programmes. Of equal importance is the ability to compare physiological and biochemical processes of cells and protoplasts selected for stress tolerance against unselected (wild-type) lines and to relate this to genetic regulation at the molecular level.

The techniques of tissue and protoplast culture are important for application of molecular biology to genetic manipulation of plants. The field of plant pest-host interactions has been significantly advanced by the use of protoplasts and subsequent tissue culture and regeneration of pest-resistant plants (Harrison & Mayo, 1983). Somaclonal variation has provided important genetic material both for genetic studies and for selection of desired traits in plants (Scowcroft, Larkin & Brettell, 1983; Maddock & Semple, 1986).

Introduction: improvement of drought resistance in conventional breeding programmes

Critical evaluation of progress in plant breeding over a period of several decades (e.g. Wilcox et al., 1979; Castleberry, Crum & Krull, 1984) has demonstrated a genetic improvement in yield under both favourable and stress conditions. The yield improvement under drought stress occurred before many of the physiological issues of drought resistance were understood and resulted partly from the genetic improvement of yield potential and partly from the improvement of stress resistance. For example, Bidinger et al. (1982) found that the yield of millet varieties under drought stress was largely explained by their yield potential and growth duration. Early varieties with a high yield potential were most likely to yield best under stress. Fischer & Maurer (1978) also recognised the effect of potential yield on yield performance of wheat under drought stress and proposed a ‘susceptibility index’ (5) which estimated the relative susceptibility of a variety to drought stress. In analysing their wheat data, they found that S was not totally independent of the potential yield of the variety.

The improvement of yield under stress must therefore combine a reasonably high yield potential (Blum, 1983) with specific plant factors which would buffer yield against a severe reduction under stress. On the other hand, potentially lower yielding genotypes occasionally have been found to perform very well under drought stress conditions (e.g. Blum, 1982; Ceccarelli, 1987), especially under severe drought stress. One is left with the long-standing practical conclusion of Reitz (1974) that ‘Varieties fall into three categories: (a) those with uniform superiority over all environments; (b) those relatively better in poor environments; and (c) those relatively better in favoured environments’.

Introduction

The study of agricultural microbiology has expanded in recent years and one of the areas of particular interest has been biological control. The aim of this book is to provide an introduction for undergraduate students, or for research workers in related subjects, to the biological control of plant pathogens, especially of agricultural crops. The subject overlaps with the study of microbial inoculants that may be commercially available (e.g. Rhizobium) or still in the development stage like those for some mycorrhizas. Indeed the problem with biological control is that it impinges on so many other subjects because the approach is always holistic: it tries to combine the manipulation of edaphic and microclimatic factors with crop husbandry, plant breeding and direct intervention with microbial inoculants to produce maximum plant growth and minimum disease. This practical, commercial bias with agricultural crops has become dominant, but biological control does of course originate in natural ecosystems where in general serious disease is the exception and pathogens are supposed to have co-evolved and to exist in balance with their higher plant host and with the other microorganisms in their environment.

Biological ways of controlling disease have therefore existed for as long as hosts and plant pathogens, and they have been the only way of disease limitation until the last few years when chemicals became available. Lime sulphur was introduced in 1802 and Bordeaux mixture in 1882. This very recent advent of pesticides, like many other new ‘fashions’, has led to a temporary over-reaction and their over-use in some situations.

Introduction

There are possibly two major reasons why stress physiology is currently the subject of extensive research. The first is the value to the exploration of basic plant science of perturbations in relatively simple edaphic and climatic factors such as temperature, aeration, external osmotic pressure and specific salts. The second is the recognition within the international community, both in the rich donor countries and in the poor recipients of that aid, that poor and possibly declining environmental conditions and, more crucially, the amplitude and unpredictability of climatic changes and chronic edaphic conditions, are major threats to the welfare of much of the human race. Therefore understanding and exploiting the resistance of some plants to environmental factors such as drought, waterlogging, high and low temperatures, and salinity, are regarded not simply as physiological or ecological problems, but increasingly as important goals of international economic, political and humanitarian significance.

Use and misuse of the term stress has been discussed in detail in Chapter 1. In this review we explore the use of comparative cell physiology and biochemistry, concentrating on the analysis of drought and salinity and the cellular responses to these stresses, as a means of gaining an insight into the underlying processes and of diminishing our dependence on an anthropomorphic concept of stress as deviation from an agronomically-conceived norm.

Growth, membranes and cell walls

In plants volumetric growth is primarily the result of cell expansion by the development of a large vacuole. Both growth and the mechanical rigidity of tissues require that a substantial turgor pressure is sustained.

Introduction

The main diseases that we are concerned with in this chapter are seed rotting, pre- and post-emergent damping off and various seedling blights. This complex of diseases is mostly caused by a few genera of fungi, especially Rhizoctonia and Pythium, with Phytophthora, Fusarium and Sclerotinia causing less widespread problems. These are unspecialized pathogens (r-strategists) which use exudates from the germinating seeds for saprotrophic growth before they attack the very young plants that have not developed effective mechanical barriers to infection. Rhizoctonia usually attacks the seed, hypocotyl or stem, while Pythium attacks the root tips. Under very wet conditions Rhizoctonia and Phytophthora may grow amongst the tops of the seedlings. The diseases are especially bad if conditions are not favourable to the rapid growth of the seedling. Damping off is characteristic of crops that have been sown too early in the year so that they are germinating slowly in damp soils at low temperatures. Vigorous seedlings getting away to a good start under ideal conditions do not usually suffer from these problems.

However, often there are agricultural reasons for trying to make an early start to the growing season, or seeds sown in a warm period of weather may be overtaken by a cold wet spell. In glasshouses, where many seedlings are raised for horticultural crops, there are less climatic problems, but heating costs are high and some growers may try to manage with just a little less heat than is best, or seeds may be over-watered. Seed rots and damping-off remain a real problem.

Introduction

The use of stress terminology has been discussed in Chapter 1, where it was pointed out that the value of the term stress in indicating some adverse force or influence lies in its extreme generality, without the need for a precise quantification. Nevertheless it is appropriate that a scientific discipline should be concerned with definable quantities. This will be the starting point for this paper, which will follow the example of Levitt (1972) who applied the concepts and terminology of mechanical stress (force per unit area) and strain (a definable dimension change) to the study of plant responses to the environment. This approach will be developed here in an attempt to incorporate the philosophies behind stress effects into a general treatment of the responses of ecosystems to adverse environmental conditions.

Mechanical stress

The mechanical concepts of stress are outlined in Fig. 1, with the axes reversed from that employed by mechanical engineers. The three salient features of a stress-strain response curve are shown in Fig. la. Initial increases in stress cause small strains but beyond a threshold, the yield stress, increasing stress causes ever increasing strains until the ultimate stress, at which point fracture occurs. The concept of the yield stress is more clearly realised when material is subjected to a stress and then relaxed to zero stress (Fig. lb). In this case a strain is developed but is reversed perfectly – elastically – to zero strain at zero stress. In contrast, when the applied stress exceeds the yield stress (Fig. lc) and the stress relaxes to zero, the strain does not return to zero.

Introduction

Plants are constantly subject to adverse environmental conditions such as drought, flooding, extreme temperatures, excessive salts, heavy metals, high-intensity irradiation and infection by pathogenic agents. Because of their immobility, plants have to make necessary metabolic and structural adjustments to cope with the stress conditions. To this end, the expression of the genetic programme in plants is altered by the stress stimuli to induce and/or suppress the production of specific proteins which are either structural proteins or enzymes for specific metabolic pathways.

Several problems are addressed in the study of stress-induced proteins: (1) Perception: how does a plant recognise the existence of a stressful condition? (2) Regulation of gene expression: how does the perceived stress signal alter the expression of genes? (3) Function: what are the physiological roles of the stress-induced proteins? Studies designed to answer these questions usually begin with the finding of new proteins in stressed tissues, most likely by gel electrophoretic techniques. This initial observation is followed by purification of the stress proteins, and the cloning and characterisation of their genes. Research on the function of stress proteins has been progressing, although many stress proteins remain unidentified. The least understood process is probably the molecular mechanism underlying the perception of stress signals.

Temperature stress

Due to seasonal changes, almost all plants are affected by temperature fluctuations in their life cycles. Very high temperatures have been reported in many arid zones around the world, and the lack of effective transpiration in plants located in these areas causes the temperatures inside these plants to be significantly higher than ambient (Levitt, 1980). Chilling or subfreezing temperatures are even more common.

This book is a response to the increasing interest in biological control of plant diseases which is being shown by academic, commercial and agricultural organizations, and individuals, all over the world. The subject now receives more than a brief mention in most courses taught on plant pathology and is clearly going to be more important in the future. The excellent books by R. J. Cook and K. F. Baker will remain the standard references and there are now many review articles on various aspects in specialist journals. The present text attempts to provide an account of biological control of plant diseases that will be suitable for undergraduate students at college or university who will be meeting the subject for the first time. It is hoped that teachers at other levels will find it useful and that it will help research workers in many fields to enter the literature on disease control through biological means. The two introductory chapters attempt to set out general principles of microbial, host and pathogen interactions, and the historical and commercial background to biological control. The glossary is not comprehensive, but is designed to help those with a limited background in plant pathology and ecology.

There is a great deal of information produced for and by the agricultural industry on the chemical means of controlling diseases, as well as a vast research and teaching literature. Hopefully this book will provide some readily available information and examples of biocontrol. I by no means disparage the enormous benefits that have resulted from the correct use of pesticides, especially fungicides so far as we are concerned, but there is now a need to present a balanced argument for and against different methods of disease control.

Introduction

There is much information on the microbiology of soils (Nedwell & Gray, 1987), especially the soil near roots which will mostly concern us. Soils are very variable on many different scales. There are the differences that occur between soil types, usually based on the parent material, the climate and the vegetation, which control the amount of clay, organic matter and so on. Secondly, there are differences within soil in relation to depth, which reflect the addition of organic matter to the surface and are the result of leaching down the profile: the soil may be divided into a number of layers (called horizons) which have very different physical and chemical characteristics. Thirdly, there are differences on a very small scale, the microhabitat, which reflect changes in nutrient status, substrate availability, aeration, etc. on different parts of a soil crumb or a sand grain: we may here be talking about distances of a few tens of micrometres making a significant difference in oxygen levels because of the very low solubility of this gas in water (Campbell, 1983; Bruehl, 1987). There is, therefore, great variation in microbial numbers and activity between and within soils. There may be several million bacteria and hundreds of thousands of fungi which can be cultured from a gram of soil, but many of these will be inactive in the soil because of the environmental limitations which most commonly are temperature, water availability, aeration and available substrates for metabolism and growth.