SIGNS AND SYMPTOMS
In human medicine the patient presents two sets of information to the doctor: the ‘signs’ and the ‘symptoms’ of the illness. Some of the signs are obvious when the patient walks over the threshold limping, for example, or clutching an arm or a leg. Other signs are revealed when specific tests are carried out with the stethoscope or by chemical and microbiological techniques. The symptoms are revealed when the patient describes, for example, feeling nauseous, breathless or lethargic. The diagnosis of the illness is made by a careful analysis of this information, checking for particular headmarks and rigorously excluding solutions which do not fit the expected range of signs and symptoms.
EXAMINING THE PLANT
Plants are much less complex than humans. They cannot speak, however, so the task of diagnosing plant disease is harder (the cynic might say easier). The ‘symptoms’, here defined as the host response, such as yellowing, wilting or root death, are valuable pointers to the nature of the disease. Time should, therefore, always be allowed for a considered appraisal of the diseased plant and a comparison with healthy neighbouring plants of the same and of a different species: is the condition confined to one species, is there evidence of weed-killer damage, a previous fire, flooding, or a shortage or over-supply of specific nutrient chemicals? Once it is established from the symptoms that there is a disease condition, by far the greatest attention is paid to the signs: the presence of a specific organism, its morphology and its identity. Plant pathologists use the terms ‘signs’ and ‘symptoms’ very loosely and most often refer to symptoms to embrace both sets of information.
GETTING STARTED
To begin the study of plant diseases, a keen eye is required, to spot the signs and symptoms of disease (see Waller et al, 1998) and a sharp knife to cut off small pieces of diseased tissue for closer examination or a small trowel to expose the roots, if infection below ground is suspected. Since good records are essential in plant pathology, also needed are a camera (with a close-up lens) and pencil and notebook (or an electronic equivalent), to record the observations. A basic library of reference books is necessary to help identify the diseases and their causal pathogens (see Bibliography). Since, as already suggested, lesions and blemishes on plants are not always caused by a pathogen but may result from nutrient deficiency or toxicity, an appropriate reference book (Bennett, 1993) is included in the Bibliography.
EXAMINATION WITH THE NAKED EYE AND HAND LENS
The first examination is with the naked eye. Indeed, the naturalist trains his or her eye to recognise unusual colours, shapes or architecture in the natural plant community, and distortions or spots on individual leaves. When something unusual is found the next step is to examine it more closely with a hand lens. This may be purchased in most photographic or optical shops. Useful magnifications are x10, x15 and x20 and the lens should be one which minimises distortions and colour changes. More detail is revealed by the highest magnification, but the greater the magnification the more difficult it is to position the eye, the lens and the specimen to achieve best focus. The x15 magnification is a convenient compromise. The lens should be positioned so that sufficient light reaches the surface of the specimen, unshaded by the observer’s head or hand. Naturalists who wear glasses may find it useful to attach them to a halter so that they can be removed and replaced easily and safely. It is not impossible to observe through a magnifying glass while wearing glasses and this is necessary for those with particular eye problems, but the additional lens makes manipulation and focusing more difficult. It is also convenient to attach the magnifier to a cord and carry it at all times.
Most plant diseases likely to be encountered by the field naturalist are caused by fungi and their spores and surface mycelium can usually be easily seen with the naked eye or hand lens. Bacteria and viruses are too small to be seen in this way, but much can still be learned by close examination of the diseased specimen. Further guidance for the study of bacterial diseases is given by Bradbury (2000) and Lelliot and Stead (1987), and for virus diseases by Hill (1984), Walkey (1990) and Matthews (1991, 1992).
EXAMINATION WITH A MICROSCOPE
More detailed examination may be carried out by using a stereoscopic (low power: x20 – x200) dissecting microscope or a compound (high power: x100 – x1000+) microscope. The naturalist can do quite a lot without a microscope, but there is no doubt that possession of such a piece of equipment adds greatly to the enjoyment, precision of examination and understanding of the material. Stereoscopic microscopes have a considerable depth of focus and are relatively easy to use. However, the magnification is insufficient for the identification of smaller fungal spores and structures or bacteria and viruses. Compound microscopes give excellent magnification, but ideally the specimen must be mounted on a slide and specially prepared for examination by, for example, clearing, squashing or sectioning, and often staining. Appropriately prepared bacteria are visible at higher magnifications, but viruses are beyond the reach of even the best light microscopes. We suggest that you consult your local optical equipment supplier for advice on the different kinds of microscope available, and the range of prices. Do not rush into the purchase of such an expensive piece of equipment without careful consideration. Microscopes can usually be fitted with a camera to record selected images, but purchase costs are relatively high. Excellent advice on the use of microscopes and the preparation of specimens for examination is given by Waller et al (1998), Hawksworth et al (1995) and Johnston and Booth (1983). Most suppliers also provide instruction booklets.
Simple microscopic examinations can be made with the minimum of knowledge and a little trial and error. (Hazard: artificial light must not be too powerful and should be diffused through a filter to protect the eye. Similarly, the very greatest care should be taken if daylight is used as the light source.) A device to allow the substitution of filters is useful to bring out contrasts in stained material. From the point of view of safety, it is essential to study the instructions supplied with the microscope before embarking on an examination.
The identification of fungal and bacterial pathogens frequently requires that the cells or spores are carefully measured. This is achieved using a graduated eyepiece micrometer on the microscope, calibrated using a micrometer slide. These may be purchased at the same time as the microscope.
HANDLING THE MATERIAL
Material collected and brought back for examination should be processed as soon as possible. Good preparations can usually be made by removing a small portion of the infected material, transferring it to a drop of tap water on a microscope slide and repeatedly teasing it into tiny fragments with a pair of dissecting needles. These can be purchased from laboratory suppliers but adequate tools can easily be made by cutting off portions of sycamore or hazel twigs (10–15 cm long) of convenient thickness and pushing the base of a large pointed needle into the centre of the twig with a pair of pliers. The grip of wood on metal increases as the twig dries out. With a packet of mixed sewing needles a range of dissecting needle sizes may be manufactured.
When the material has been well teased out a few of the smaller portions with fungal attachments should be transferred to a fresh drop of water using a glass pipette, covered with a cover glass and gently tapped for about 30 seconds with the handle of the needle, a pencil or other suitable object. The scattered fragments should then be scanned under the low power of the microscope. When suitable spores, conidiophores or portions of fruiting bodies are found they may be examined in more detail under the higher powers of the microscope. The thickness of the coverglass is important. Too thin and it will be delicate to work with, too thick and it will impede the search at higher magnifications (size No 1.5, 22 x 22 mm square is a useful compromise).
A great deal can be discovered using simple water mounts, but for more sophisticated analysis materials need to be chemically fixed and stained with, for example, lactophenol containing 0.01% cotton or trypan blue. Details of the procedures involved for this and other fixatives and stains are given by Waller et al (1998) and Johnston and Booth (1983). (Hazard: take great care in using fixatives and stains, for many of the reagents are toxic or carcinogenic to humans.)
SECTIONING MATERIAL
While satisfactory examinations can be made by teasing out infected material, a better view of the relationship between a fungus and the host tissues, and often of the structure and morphology of the fungus, may be obtained in a transverse section of the infected stem, root or leaf. An older generation of botanists wielded ‘cut throat’ razors to produce sections of material held in the hand, often with spectacular results. Nowadays such razors are hard to obtain and recourse is made to safety razor blades backed with a stiff metal strip, sold for cosmetic purposes, or sharp disposable scalpels obtained from medical instrument or graphic materials suppliers.
If the material to be sectioned is delicate it should be placed in a cleft cut in a piece of carrot tissue, or better in a piece of pith of the elder (Sambucus nigra, not the red-berried elder Sambucus racemosa). The pith is gathered from recently suppressed twigs and obtained by stripping away the thin bark and wood. The cylinders of pith are stored in dilute alcohol (25–50% surgical or methylated spirit). Sectioning involves cutting a cleft in the pith, trimming the piece of stem or leaf to an appropriate size for incorporation therein and then slicing off thin sections of pith and specimen. The top of the pith and the enclosed material should be moistened with a drop of water, and the blade dipped in water. Then, with the pith and specimen grasped firmly between thumb and curled index finger of the left hand, and the blade grasped in the right hand (or vice versa) a series of horizontal cuts is made into the pith and enclosed specimen, striving all the time to cut as thinly as possible. (Hazard: take great care in carrying out such operations, for it is easy to slip and cause injury). It is not necessary, indeed it is counter productive, to try to produce a complete transverse section; a series of thin fragments is much more useful. The fragments should be removed from the blade using a small moistened paintbrush and mounted in a drop of water on a slide, for preliminary assessment. Fragments showing interesting features may then be transferred to a separate slide for closer examination. If it is useful to substitute a staining material for the water mount without disturbing the fragment of infected plant material, a drop of the stain (see Waller et al, 1998) may be placed on one side of the coverslip in contact with the water in the mount and drawn under the coverslip by applying a piece of blotting or filter paper to the opposite side.
PRESERVING MATERIAL
The foundation of the study of natural history is recording. As Captain Cuttle said in Dickens’ Dombey and Son, “When found, make a note of!” With plant diseases, not only is it necessary to write down details of the specimen or phenomenon observed, it is also especially helpful to keep specimens of the diseases and pathogens for later examination and comparison.
After preliminary examination and approximate identification of the fresh material, herbaceous specimens and tree leaves may be dried in an herbarium press. This may be purchased or easily manufactured from two wooden frames formed of upright and cross pieces of lath to form a lattice of right-angled squares, the whole frame approximately 45 x 60 cm. Between these two frames pieces of dry newspaper cut to the appropriate size are stacked and the whole held together with a webbing strap. Specimens for drying are placed between the papers along with a note of the place and time of collection and any preliminary identification or notes on appearance, and so on. Drying is faster under warm conditions and the appearance of the specimens is enhanced if pressure is applied to the press using a heavy weight.
Specimens of wood or bark should not be pressed but dried directly in a warm atmosphere.
Smaller specimens (the majority unless one is making a special study of tree diseases) may be stored in paper envelopes folded to a standard pattern from sheets of A4 paper. These store easily in card index boxes alphabetically, or they may be numbered. It is useful to make special classifications in pursuit of a research project. Thus rusts on thistles, for example, could be classified by host, by location or by altitude or latitude to unravel their complex taxonomy. Larger specimens may be stored in A4 envelopes and kept together in a box file, and the very large woody specimens may be kept in cardboard boxes.
Specimens may deteriorate in storage from attacks of herbarium beetle. Another small beetle commonly destroys smuts and leaves behind packets of digested spores (to the confusion of the novice). To overcome such problems various strategies may be used. For example, the dried specimens may be placed in a deep freeze at -25°C before being stored. If the problem persists resort must be made to insecticides. (Hazard: most insecticides are toxic to humans and must therefore be used only in accordance with the manufacturer’s instructions.) In the case of a large collection it is worth consulting the nearest herbarium (such as at the Royal Botanic Gardens, Kew, the Royal Botanic Garden Edinburgh or the International Mycological Institute, Egham, UK) for details of the insect control protocols which they observe.
IMPORTANT CONSERVATION NOTE
The collection of large samples or whole plants from the field, whether diseased or not, must be carried out with due regard for the conservation status of the host, the pathogen and the habitat (see Ingram, 1999). Plants should not, therefore, be collected from the field without the advice of a national herbarium such as one of those listed above, a local natural history society or museum, or an appropriate national society (see here).
HANDLING THE DRIED MATERIAL
When dried material is removed from the collection for examination it is dry and full of air sucked into the drying tissues. It is also extremely precious because the chances are that there will not be the opportunity to collect exactly the same material in the same place again. Therefore only very small portions of the material should be removed for examination. These are placed on a slide in water or glycerine or lactophenol (see Waller et al, 1998) and allowed to swell. The swelling and the dispersal of air bubbles in water mounts may be hastened by gentle heating over a flame, but care must be taken not to distort the material.
INOCULATING LIVING PLANTS
The inoculation of plants with a suspected pathogen cannot be done on the spur of the moment, for the seed of the appropriate host must be collected or purchased, stored and grown under correct conditions. Nature is a good guide here, and seed of wild plants (but see Conservation Note) collected in the autumn can often be sown direct and exposed to winter temperatures. Germinated seedlings are pricked out into pots and grown on in a cool greenhouse or frame.
Inoculation may be achieved in the case of some fungi and bacteria by suspending or laying the infected material on top of the seedlings. With some pathogens spores can be shaken off the infected specimen and with some rusts and powdery mildews the spores may be removed with a knife or brush and placed on the experimental seedlings. Once inoculated the seedlings must be kept free from further contamination and kept in a moist atmosphere (they may be lightly sprayed before or after inoculation, although the presence of free water can inhibit infection by fungi such as powdery mildews). Good conditions for infection may be achieved using bell-jars made from plastic soft drinks bottles, with the bases cut off. The cap may be left in place or substituted by a wad of cotton wool. The soil should be moist and the surrounding staging should be kept well watered. It is essential also to keep an equal number of healthy, uninoculated seedlings treated in exactly the same way as the experimental plants to act as controls.
Inoculation of plants with viruses requires a large number of healthy seedlings grown in a glasshouse under insect-free conditions, to prevent contamination from outside sources. The simplest procedure with the commoner sap-transmissible viruses is to grind up the suspected diseased plant and to rub the juice onto the leaves of the plants to be inoculated. The efficiency of the process is aided by the use of a chemical buffer and the addition of an inert abrasive such as fine carborundum powder to the juice and, after inoculation, by washing the surface of the host leaf with fresh water. Refinements of this technique and methods for the inoculation of plants using insect and other-vectors are dealt with by Walkey (1990) and Hill (1984).
MORE ADVANCED STUDIES
More advanced studies of fungal diseases and identification of bacteria require access to a simple laboratory in which culture media may be made up and the pathogens isolated from diseased tissue and grown on in aseptic conditions (i.e. in the absence of other contaminating organisms), at a constant temperature. Excellent advice on setting up such a laboratory, making media and culturing pathogenic fungi is given by Waller et al (1998) and Smith and Onions (1994). The recipes for many culture media for fungi and bacteria are also listed by Johnston and Booth (1983). Methods for use with bacteria are also discussed by Waller et al (1998) and Bradbury (2000), and are described in detail by Lelliot and Stead (1987).
Viruses are normally identified using immunological or molecular biological techniques and powerful electron microscopes. The use of these normally requires access to a professional virus research laboratory, although immunological tests for some viruses of crop plants are now available in kit form (Hill, 1984; Walkey, 1990; Matthews, 1992).
Modern assays for the identification of fungi may also rely on immunological or molecular biological techniques (e.g. Schots, Dewey and Oliver, 1994) and again access to a professional laboratory is essential for their use.
KOCH’S POSTULATES
If a specific pathogen is to be unequivocally identified as the cause of a particular disease, Koch’s postulates must be fulfilled. These may be summarised as follows (after Waller et al, 1998).
i) The pathogen must be consistently associated with the disease.
ii) The pathogen must be isolated and grown in pure culture, and its characteristics described. In the case of obligate parasites, which are incapable of growth in culture, the pathogen must be grown in isolation on a susceptible host plant and its appearance and effects noted.
iii) The pathogen from pure culture must be inoculated onto healthy plants of the same species or variety as those on which the disease was first noted, and it must produce the same disease on the inoculated plants.
iv) The pathogen must be re-isolated into pure culture and its characteristics must be exactly the same as those observed in postulate (ii).
For most routine purposes it is necessary only to note that a putative pathogen is consistently associated with the signs and symptoms of a particular disease and to identify it in an appropriate reference book. However, if a new pathogen is to be described or if a new host for an existing pathogen is suspected, all of Koch’s postulates must be satisfied before the discovery can be verified, recorded officially in an appropriate journal and a specimen deposited in an internationally recognised reference collection (see Waller et al, 1998; Hall and Winter, 1994).
JOINING A SOCIETY
The enjoyment of studying plant diseases is much enhanced by joining a local natural history society or a specialist national society such as the British Society for Plant Pathology or the British Mycological Society. It then becomes possible to share one’s enthusiasm and benefit from the knowledge of others. Moreover, membership of a society usually provides access to a journal or bulletin for reading about plant diseases or for publishing one’s own observations.
VALEDICTION
The simple techniques described above will carry the reader forward to seek out and develop further ways of studying plant diseases. Techniques are important, but so too are observation and thought. The question “Why?” is always with the naturalist, and observation will often give an answer, or at least allow the development of an hypothesis for experimental testing.