The problems of plant diseases have assumed alarming dimensions with changing pattern of climate change. Consequently, a thorough knowledge of appropriate plant disease diagnosis, exact cause of the disease and understanding the dynamics of disease etc. becomes very important.
This is possible only by getting proper and relevant information on experimental methodology to study the pathogen and its biology. The phytopathological techniques focus on various techniques and their application in plant pathology.
Two useful stains used for fungi with hyaline structures are cotton blue (tryptophane) and lacto-fuchsin. Cotton blue stains the chitin present in the cell walls of fungi and lacto-fuchsin also binds well to fungi cell walls and provide blue and red colour, respectively.
A high-power compound microscope providing magnifications up to 1000 times is essential for examining the morphology of bacteria and fungi. The best resolution of the light microscope is 0.2 µm or 200 nm. The bright field microscope or light microscope or compound microscope is the most common and the most indispensable instrument used in plant pathological laboratories. The most common sub-stage condenser lens is the Abbe’s condenser (1.30na) that utilizes only two lenses.
The light microscope uses light as its source of illumination and may permit four types of microscopy (bright field, dark field, fluorescent and phase contrast). While the electron microscope uses a beam of electrons instead of light waves to produce an image.
The resolution of a SEM is 3-6 nm, which is almost 100 times better than the light microscope. The SEM is useful for examining the surface structures of fungal spores. Unlike the compound microscope, which uses light to view the sample, the scanning electron microscope (SEM) uses electrons to generate an image.
Phase contrast microscope is the ideal instrument for observation of living cells and other transparent microbes without staining.
Plant pathological specimens observed under a fluorescent microscope are coated with fluorescent compounds (Flurochrome) such as Auramine O, Acridine orange. Fluroscein etc.
‘Laminar flow’ biological safety cabinet employing high efficiency particulate air (HEPA) filters, which remove 99.97% of 0.3µm particles, is one of the most important filtration system.
The adhesive tape technique is particularly useful for showing the orientation of dry spores (as well as spore chains and the shape of conidiophores).
A freezing microtome is especially useful for cutting thin to semi-thin sections of fresh, frozen tissue.
Gram staining is useful to differentiate between two biochemically different bacterial groups, gram-positive and gram-negative (gram-positive cells stain dark purple, while gram-negative cells stain red). In Gram’s staining, iodine acts as a mordent (fixative) to form an insoluble complex with crystal violet inside the cell.
The immersion time for sterilization of any tissue infected with fungal infection in any solution is usually 1-5 minutes. Most plant pathogenic fungi grow well at 25°C. Pure cultures can be obtained from the primary isolation plates by colonies initiated from single spores or from hyphal tips.
King’s B agar is a good medium for general isolation of plant pathogenic bacteria, particularly fluorescent pseudomonads and Erwinia spp. V-8 juice agar (V-8J) is a good medium for the growth of fungi, particularly Phytophthora.
Nutrient agar is a good general-purpose medium for bacteria. Glycerol agar (GA) is used to preserve dried agar cultures as herbarium specimens. The culture is covered with mineral oil to the depth of 1 cm above the top of the short agar slope.
Soil storage method is generally used to store Fusarium spp, which often mutate if maintained on agar for long periods of time. Silica gel storage is suitable for organisms that survive freeze-drying, including mycelial forms that produce sclerotia or chlamydospores.
Except an isolate of Puccinia graminis f.sp. tritici, Gymnosporangium juniperi virginianae and Uromyces ari-triphylli, no other obligate parasite appears to have been cultured on synthetic media.
Media should have a pH range from 6.0-6.5 for fungi, as fungi usually grow best in slightly acidic medium, whereas for bacteria, it should be neutral pH 7. Carbohydrate rich medium usually encourages good vegetative growth but not favorable for reproduction.
Molecular membrane filters are cellulose-plastic porous membranes available in different pore size grades from 5µ-10µ. Suitable dilutions for isolation of plant pathogens from soil are 10-3 to 10-4 for actinomycetes, 10-5 to 10-6, and 10-4 to 10-5 for fungi.
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