Introduction

The crucial role played by the neutrophil in protecting the host against bacterial and fungal infections is highlighted in patients with defects in neutrophil function. These individuals, either with low numbers of circulating neutrophils (i.e. neutropenias) or with specific defects in one or more key processes, are predisposed to life-threatening infections. Indeed, at neutrophil counts of < 1000/μl blood, individuals are at risk from infections, with the risk of infection inversely proportional to the neutrophil count. Neutropenias may be due to disease, such as congenital neutropenia, aplastic anaemias, agranulocytosis, acute myeloid leukaemia and the myelodysplastic syndromes. In such cases, CSFs (e.g. GM-CSF, G-CSF) have been used clinically to improve the circulating neutrophil count. Whilst initial results have shown some promise, such CSF therapy is not without its drawbacks. Some malignant cells may be stimulated by CSF therapy, and accumulation of neutrophils in the lungs may occur. Circulating levels of CSFs may also increase the expression of adhesion molecules on the surface of circulating cells, which may result in increased adhesion of neutrophils to endothelial cells or in increased neutrophil–neutrophil or –platelet interactions.

Neutropenias may also arise as a side effect or deliberate consequence of therapy. For example, some drugs used in the treatment of inflammatory disorders are immunosuppressive, and if these decrease the number of circulating neutrophils to below the critical threshold level, then susceptibility to infection may result. During chemotherapy for the treatment of solid tumours, an inevitable consequence of cytotoxic therapy is that the bone marrow will be destroyed by the drugs; thus, patients will have a considerable risk of infection during this induction period.

Haematopoiesis

The process responsible for the production of blood cells is known as haematopoiesis, which occurs in the bone marrow. In an adult human, the bone marrow weighs about 2.6 kg, which accounts for 4.5% of the total body weight. Although the bone marrow is dispersed throughout the body, it is nevertheless a larger organ than the liver, which weighs about 1.5 kg. About 55–60% of all cells produced by the bone marrow are neutrophils. The marrow is thus highly proliferative, with mitoses observed in 1–2.5% of all nucleated cells. The cellularity of the marrow varies considerably with age: for example, in the young about 75% of the marrow comprises cells, whereas in adults this figure is decreased to about 50% and in the elderly only 25% of the marrow is cellular.

The bone marrow

The bone marrow is comprised of the cells that divide and develop into mature blood cells and also of stromal cells consisting of fibroblasts, macro – phages and adipocytes. There are three major cellular types in the marrow (Fig. 2.1); these give rise, by the processes of division and differentiation, to the eight major blood cells.

1. i. Pluripotent or multipotent stem cells have the ability to divide and differentiate into blood cells of all lineages, but are also capable of selfrenewal. Thus, a pluripotent stem cell can differentiate to form two cells that are more mature (i.e. myeloid- or lymphocyte-like), or else it can divide to form two identical, uncommitted stem cells. Indeed, experiments with irradiated mice have shown that as few as 30 stem cells can replace all of the cells of the bone marrow.

2. […]

The cytoplasm of the neutrophil is highly structured and is organised into four distinct components that constitute the fibrillar meshwork: the microfilaments, microtubules, intermediate filaments and the microtrabecular lattice. These components form the cytoskeleton, the supporting framework for the cell, within which the intracellular components are embedded. Thus, the translocation of cytoplasmic organelles and granules, the movement of cytosolic proteins, the recycling of receptors – as well as key neutrophil functions such as cell movement, phagocytosis, NADPH oxidase activation, degranulation and receptor regulation – are all intimately associated with changes in the organisation of the cytoskeleton. Therefore, in order for the neutrophil to be able to mount the appropriate response upon exposure to stimuli, plasma membrane occupancy must be linked to changes in cytoskeletal organisation via the generation of second messengers. Furthermore, because activation of some neutrophil functions or changes in morphology may be observed within seconds of an agonist binding to its receptor, the cytoskeleton must be capable of responding very rapidly to the production of such second-messenger molecules. It is also now becoming accepted that cytoskeletal reorganisations may also terminate some responses – as in the termination of second-messenger production by interaction of cytoskeletal elements with receptors to down-regulate their function, or by receptor internalisation. Thus, the cytoskeleton is a highly-complex network comprising numerous proteins, and these respond to second messengers to undergo rapid changes in molecular reorganisation upon appropriate stimulation.

Introduction

Whilst the neutrophil has one primary function – namley, to recognise and destroy microbial pathogens – many separate but interrelated processes are required to control this function. For example, the neutrophil must be able to detect a variety of pro- and anti-inflammatory signals in its microenvironment and then respond accordingly. The responses to these signals include up-regulation of receptor number and function, adherence, diapedesis, chemotaxis, particle binding, phagocytosis, degranulation and activation of the NADPH oxidase. There may also be, where appropriate, the generation of further pro-inflammatory mediators, such as eicosanoids and secondary cytokines. Furthermore, these responses must be terminated when the noxious agent or pathogen has been eliminated. Such deactivation mechanisms of neutrophil function are almost as important as the activation mechanisms, because prolonged or inappropriate neutrophil activity can lead to inflammatory damage via the secretion or overproduction of cytotoxic products.

All known physiological and pathological agents activate neutrophils by first binding to plasma membrane receptors, details of which are given in Chapter 3. The cells must respond either to soluble agonists, which generally signal events such as changes in receptor expression, adherence, aggregation and chemotaxis, or else to particulate stimuli (e.g. opsonised bacteria and immune complexes), which generally stimulate phagocytosis. However, some soluble agonists (e.g. fMet-Leu-Phe, C5a and LTB4) stimulate adherence and chemotaxis (which are early events in the inflammatory response of neutrophils) at low concentrations, but at higher concentrations can activate degranulation and reactive oxidant production (which are later functional responses).

Background

By the late 1970s, the idea that mature, bloodstream neutrophils were terminally differentiated, end-of-line cells was being questioned. In a series of experiments by McCall and colleagues (1973, 1979), the biochemical properties of ‘toxic’ neutrophils – that is, neutrophils isolated from the bloodstream of patients with acute bacterial infections -were characterised. Compared to normal neutrophils isolated from healthy controls, these ‘toxic’ neutrophils exhibited increased oxidative metabolism, phagocytosis, chemotaxis and 2-deoxyglucose transport. They also had increased cellular levels of alkaline phosphatase and showed toxic granulation. Two possibilities could account for these enhanced functions: either the bacterial infection caused the release from the bone marrow of a more active neutrophil population, or the infection somehow changed the activity of the circulating cells. If the latter explanation was the case, then the concept that neutrophils were terminally differentiated was probably incorrect.

At around this time, the synthetic formylated oligopeptides, thought to be analogous to bacterial-derived products, were being characterised. Remarkably, it was shown that the pretreatment of normal neutrophils in vitro with 2 × 10−8 M fMet-Leu-Phe for 15 min could alter the function of these cells so that they resembled ‘toxic’ neutrophils. The conclusion, therefore, was that factors induced by the bacterial infection (either derived from the bacteria themselves or else generated by the host in response to the infection) could up-regulate the function of mature, circulating neutrophils.

It was later shown that substimulatory concentrations of many types of neutrophil agonists could also induce this up-regulation of function. Because the concentrations of these compounds that were required did not activate the neutrophil per se, the phenomenon was referred to as ‘priming’.

Plant pathogenic bacteria are typically motile, single-celled organisms, for which a range of light and electron microscope techniques are available to investigate aspects of structure and morphology (Sigee, 1989). Determination of the chemical and structural organisation of plant pathogenic bacteria is important to an understanding of cell function and host—pathogen interactions, and is also an important factor in bacterial taxonomy and pathogen identification. Phytopathogenic bacteria can be examined either during growth in sterile medium (in vitro culture, Fig. 2.1) or in association with higher plants, where they may be present on the plant surface or within infected tissue (growth in planta, see Fig. 2.3c).

Characteristic morphology and fine structure

The small size (0.5–2.0 μm diameter) of plant pathogenic bacteria places them close to the limits of resolution of the light microscope, and the examination of bacterial preparations with this instrument normally involves the use of an oil immersion objective to obtain maximum detail. These organisms are also quite difficult to see in terms of their optical contrast, and light microscope examination normally involves the use of stained preparations or phase-contrast microscopy (see Fig. 6.17a). Under optimal conditions of light microscopy, general features of morphology such as size, shape, and the presence of flagella and a capsule may be resolved, but little further detail can be determined.

The ability of plant pathogenic bacteria to survive and multiply outside and inside plants, and to cause disease, is determined to a large extent by their genetic constitution. The genetic analysis of plant pathogenic bacteria currently involves the application of molecular techniques for the identification and investigation of bacterial genes that are important in all of these aspects, and will be considered first. Following sections discuss the role of specific genes and gene systems in the activity of plant pathogenic bacteria in relation to the determination of compatibility and incompatibility, disease virulence, and non-pathogenic characteristics. The final part of this chapter deals with the occurrence and role of plasmids in these bacterial cells.

Molecular genetics: identification and investigation of bacterial genes

Bacterial genes, occurring on either chromosomal or plasmid DNA, are involved in the determination of a wide range of phenotypic characteristics. In recent years new techniques of molecular biology have been particularly successful in the genetic analysis of plant pathogenic bacteria (Daniels et al., 1988), and have been described in detail in a number of recent texts (e.g. Brown, 1986; Sambrook et al., 1989). The major objectives of molecular genetics are:

1. Identification and isolation (cloning) of specific genes with defined functions.

2. […]

Infection of plants by pathogenic bacteria can generally be considered in terms of three interrelated phases:

1. Population build-up, competition and migration of bacteria at the plant surface.

2. Bacterial entry into plant tissue.

3. Migration of bacteria within the plant to and from regions of multiplication.

Build-up and activity of epiphytic populations

Population level

The presence of epiphytic pathogens on host plants does not imply that disease will necessarily develop, and many cases have been reported where quite high levels of pathogenic bacteria were present on symptomless foliage. This has been noted, for example, for Pseudomonas syringae pathovars on red maple (Malvick & Moore, 1988) and snap beans (Legard & Schwartz, 1987) and for Erwinia amylovora on apple and pear blossom (see later).

In other situations, the presence of epiphytic bacteria does lead to disease development. This was initially noted by Crosse (1957), who reported the presence of Pseudomonas syringae pv. mors-prunorum as an epiphyte on cherry foliage, leading to canker formation. The relationship between epiphytic occurrence and disease development has subsequently been investigated for a wide range of bacterial pathogens by monitoring naturally occurring populations and carrying out experimental inoculations of plant surfaces. These studies have shown that plant infection and disease development depend on a number of factors, including the particular host—pathogen combination, critical environmental conditions, physiological stress of the host plant and the attainment of minimal threshold levels of the pathogen.

Although compatible phytopathogenic bacteria share a common ability to spread and multiply within the host plant, the manner in which they do this and the effect they have on the host plant (disease) vary considerably. This chapter considers general aspects of disease induction, different types of disease that are caused by plant pathogenic bacteria and the range of bacterial characteristics that are important in disease development.

The induction of bacterial disease

The ability of plant pathogenic bacteria to cause disease in a particular host plant depends on many features, including environmental aspects, plant physiology and development, and the expression of pathogenicity and virulence factors by the bacterial cells.

Environmental and physiological factors affecting disease development

Environmental factors are important in the development of plant disease for their direct effects on infection (Chapter 5) and for their indirect effects in determining the physiological status of the plant.

The various aspects of the plant which affect disease development are discussed by Lozano & Zeigler (1990) and include nutritional status, photoperiodic conditioning and stage of maturity and development.

Levels of macronutrients have been shown to be important in plant susceptibility to Erwinia stewartii, where elevated levels of N and P increase susceptibility and high levels of Ca and K increase resistance.

The taxonomy of plant pathogenic bacteria, with its three interrelated aspects of classification, nomenclature and identification, is a central aspect of bacterial plant pathology. It is clearly important to be able to establish the identity of an isolated plant pathogenic bacterium, so that the agent causing a particular disease — or with the potential to cause disease — can be clearly defined. Phytopathogenic bacteria may be isolated from a wide range of sources, including infected plant tissue, seed surfaces, soil and water environments, and the identification of bacteria from such sites has implications not only for disease diagnosis and pathogenicity but also for studies on disease epidemiology and aetiology. In addition to defining bacteria as agents of disease, taxonomic studies can also provide useful insights into phylogenetic relationships between the phytopathogens.

The taxonomy of plant pathogenic bacteria will be considered in relation to two major aspects:

1. The establishment of a clearly defined and internationally accepted system of classification and nomenclature.

2. Bacterial identification, including: isolation and identification from different sites, pathogenicity testing, in vitro diagnosis, and computer identification by numerical analysis. There is now a wide range of features on which bacterial classification can be based, and which can be used for identification.

General principles of bacterial taxonomy, with details of the various characteristics that can be used for classification, are discussed in Bergey's Manual of Systematic Bacteriology (Kreig & Holt, 1984).

In natural environments, where a particular host species occurs within mixed vegetation, the development and spread of disease is probably limited to some extent by the separation of individual plants within the area. This constraint does not apply in the crop situation, where localised infection and progression of disease within the homogeneous plant population can occur rapidly. In this artificial situation, where the natural balance between pathogen and host does not apply, special control measures often have to be adopted if the large scale occurrence of disease and consequent major crop loss are to be avoided. These measures fall into four main categories: chemical control, biological control, breeding of resistant cultivars and sanitary procedures.

Chemical control

Chemical control agents are of two main types: bactericides (synthetic organic and inorganic compounds) and antibiotics (naturally occurring microbial products). The use of these two types of control agent is considered in the first part of this section, with a final discussion on general aspects of chemical control.

Bactericides

The range of compounds used as bactericides has recently been reviewed by Sekizawa and Wakabayashi (1990) who divide these compounds into four main categories: synthetic bactericides formerly used for crop protection, currently used synthetic bactericides, traditional inorganic compounds and soil nitrification inhibitors.

Plant pathogenic bacteria are not restricted in their occurrence to infected plant tissue, but are widely dispersed throughout the external environment. This chapter will consider the general occurrence of plant pathogenic bacteria in the aerial and soil/water environments, environmental interactions at the micro-level and the association of these bacteria with invertebrates (vectors).

The aerial environment

The aerial occurrence of plant pathogenic bacteria is clearly of particular relevance to those pathogens that infect aerial parts of plants, including leaves, flowers and fruit. The aerial environment includes both physical aspects (e.g. occurrence of bacteria in rain and aerosols) and biotic aspects (occurrence of bacteria on plant surfaces and aerial dispersal by vectors).

Occurrence of bacteria in rain and aerosols

The aerial environment presents a potentially important medium for both survival and transmission of plant pathogenic bacteria, particularly where cells are contained in rain water or fine water droplet dispersions (aerosols).

Rain-water from infected foliage may contain high levels of phytopathogenic bacteria, and may be important in the spread of bacteria both within and between plants. A good example of rain dispersal of pathogen within single plants is provided by the studies of Miller on Erwinia amylovora (reported in Van der Zwet & Keil, 1979), who showed that if a source of inoculum was present in the upper part of a tree, the region of secondary infection below was cone-shaped due to downward dispersal of bacteria by rain-splash.

The entry of bacteria into the plant during the infection process leads to various types of interaction, observable at the level of the whole plant, constituent tissues or individual cells. These interactions have been investigated experimentally by artificial infiltration of intact plants (‘Inoculation of intact plants’, this page) or by the use of in vitro systems (including micropropagates, excised organs and cell suspensions; see ‘Use of in vitro systems’, p. 132).

Inoculation of intact plants

The effect of different bacteria in determining the nature of the plant response was initially demonstrated by Klement et al. (1964), who artificially infiltrated leaves of tobacco with a range of bacterial species (Fig. 6.1) and observed three main types of result:

1. Hypersensitive reaction (HR): where there is typically a rapid death of the plant cells, with no spread of bacteria to surrounding tissues. This reaction was induced by a range of bacteria comprising various pathovars of Pseudomonas syringae.

2. Disease reaction: involving a delayed host cell response, with spread of bacteria to other parts of the plant. This reaction was induced by Pseudomonas syringae pv. tabaci and resulted in wildfire disease.

3. No observable reaction, after infiltration of the saprophytic bacterium Pseudomonas fluorescens.

The results obtained by Klement et al.