PreviousAcquisition feeding
The feeding period during which an insect ingests sap containing the pathogen.
Alkaline phosphatase
An enzyme that hydrolyses certain phosphatecontaining compounds under alkaline conditions; commonly obtained from calf intestine mucosa.
Antibody
A protein formed in blood serum in response to stimulation by an antigen. Antibodies are specific for their respective antigens, and antigens and antibodies are mutually attracted.
Antibody-antigen complex
The reaction or attraction formed when reaction antigens meet their corresponding antibodies or vice versa. This strong attraction is the basis of all immunodetection systems.
Antigen
A substance, often a virus or bacterium, that stimulates production of antibodies in an animal. Specifically, the corresponding molecule to the antibody in a serological test.
Autoradiography
The technique or process of making a picture revealing the presence of radioactive material, the film being laid directly on the object to be tested. Frequently used for detection of radioactivity following hybridization, by exposing filter paper to sensitive X-ray film.
Biotin
A water-soluble vitamin of the B complex widely distributed in plant and animal tissue; it binds strongly to a glycoprotein named avidin.
Biotin derivatives of deoxyribonucleotides are incorporated into probe DNA by nick translation (see Part III). After hybridization the biotin can then be detected using streptavidin-fluorescein complexes. The streptavidin binds to the biotin by one of the strongest known biological interactions. The enzyme (usually peroxidase or alkaline phosphatase) is then reacted with its substrate which gives a coloured product; fluorescein is detected by fluorescence under light of a certain wavelength.
Biotinylated compound
A compound to which the small vitamin biotin has been attached. Antibodies, enzymes and nucleic acids can be labelled with biotin.
Biotinylated enzyme
An enzyme coupled chemically to biotin.
Biotinylated probe
A DNA probe in which certain bases have been modified by chemical coupling of biotin.
Blot
Verb: to transfer DNA, RNA or protein to an
immobilizing matrix such as DMB-paper, nitrocellulose or nylon
membranes.
Noun: the autoradiograph produced during the Southern or Northern
blotting procedure.
BRL Hybri-dot
A commercially available kit for applying small volumes of extracts to a membrane to test for the presence of viruses or viroids by hybridization.
Bud-graft inoculation
A bud, blind bud or chip bud cut from a stem of the plant or tree to be indexed and grafted to an indicator plant or tree.
Bud-union crease
A line, ridge or fold, usually discoloured as brown, yellowish-brown or reddish-brown, and formed at the bud union. It is readily observed when the outer bark is removed. Some bud-union creases are caused by pathogens and others by incompatibility of rootstocks and scions.
cDNA
Complementary DNA. The DNA complement of an RNA sequence. It is synthesized by the enzyme RNA-primed DNA polymerase or reverse transcriptase. The single-stranded DNA product of this enzyme (the reverse transcript) may be converted into the double-stranded form by DNA-primed DNA polymerase and inserted into a suitable vector to make a cDNA clone. cDNA cloning is commonly used to achieve the expression of mammalian genes in bacteria or yeast.
cDNA probe
A radioactive specific DNA sequence used to detect complementary sequences of RNA or DNA (see cDNA and Probe).
Certification programme
A programme developed by a country, state, university or research centre for ensuring that selected budwood distributed to the growers is free of graft-transmissible pathogens and that the fruit is true-to-type. These pathogen-free certified trees are usually registered, and budwood issued from these mother or foundation block trees can be used to produce additional buds in an increase block for the development of certified trees.
CF-11
A fibrous, graded cellulose powder sold by the Whatman Company.
Chip bud
A piece of bark tissue used for graft inoculation. It is used when the bark of receptor indicator plants does not slip or open up to accept a bud or blind bud.
Chromatography
The separation of mixtures of chemicals, compounds, proteins, macromolecules, etc. into their constituents or components by preferential adsorption by a solid such as a column of cellulose or by filter paper or gel.
Clone
A budline derived from a single parent source by propagation from that source.
Complementary
See cDNA. A nucleic acid sequence is said to be complementary to another if it is able to form a perfect hydrogen-bonded duplex with it, according to the Watson-Crick rules of base pairing. A viral genomic ssRNA is complementary to negative-sense ssRNA from which it is transcribed.
Conjugated molecule
As used in ELISA, antibody and enzyme proteins combined to form an enzyme-labelled antibody.
The enzyme can then be detected colorimetrically, and the colour produced will fairly precisely indicate the amount of virus present.
Corky bark
Abnormal condition of grapevine bark due to excessive cork production, which confers upon the trunk a spongy texture and a rough appearance, as with corky bark disease (see Rugose wood).
Coulure
Grapevine disorder whereby berries fail to set, yielding straggly clusters. It can be induced and/ or enhanced by viral infections (nepoviruses in particular).
DEAE cellulose column
A plastic or glass tube open at the top and fitted with a stopcock on the bottom, containing diethylaminoethyl cellulose.
Denature
To so modify (a protein) by heat, acid or alkali that it retains its primary structure but no longer has all its original properties.
Deproteination
To remove and separate proteins from other macromolecules from samples to be tested by hybridization. This is normally achieved by phenol extraction or by treating with a proteindigesting enzyme.
Dialysis
A procedure using a membrane to separate various components in solution in accordance with their ability to pass through the membrane.
Dicing
Cutting of tissue into small segments using a sharp knife or razor-blade. In the ELISA technique, leaf or bark segments are diced or cut up prior to grinding.
DNA
Deoxyribonucleic acid. Any of a class of nucleic acids that contain deoxyribose, found chiefly in the nucleus of cells. Functions in the transference of genetic characteristics and in the synthesis of protein.
DNA probe
A probe for detection of specific nucleic acid segments (see Probe).
Dot-blot
A procedure used to determine the presence and concentration of a particular RNA or DNA species. Different concentrations of the nonradioactive nucleic acids are denatured and applied as a dot to nitrocellulose paper or other support matrix. This is then hybridized with the radioactive complementary probe under study. After autoradiography, the intensities of the radioactive images formed are quantified and compared to a control series to determine the concentration of the non-radioactive molecule.
dPAGE
Denaturing polyacrylamide gel electrophoresis. Electrophoresis in a gel formed from polyacrylamide in the presence of a chemical agent such as urea (8 M) or heat; the technique minimizes the effects of the secondary and tertiary structure of the molecule on electrophoretic mobility.
Electro-blot membrane
A solid charged medium on to which a molecule is fixed as a result of electrophoresis from a source medium. This can be nitrocellulose or a charged nylon matrix.
Electro -blotting
The electrophoretic transfer of macromolecules (DNA, RNA or protein) from a gel in which they have been separated to a support matrix such as a nitrocellulose or charged nylon sheet. An alternative to the capillary transfer usually used in techniques such as Southern and Northern blotting.
Electro-elution
Removal of adsorbed material from an adsorbent by use of an electric field; or recovery of a charged molecular species by electrophoretic migration from a source medium such as a polyacrylamide gel to a liquid medium in which concentration of the species can be accomplished.
Electrophoretic techniques
Techniques of separating components suspended in a fluid media or gel by the influence of an electric field.
Electro-transfer
The movement of a charged molecule from one medium to a second by migration in an electric field.
ELISA
Enzyme-linked immunosorbent assay. Two antibody preparations are commonly used in ELISA. The primary antibody binds the antigen, which is itself bound by the second antibody. The second antibody is linked to any enzyme whose activity is easily monitored, i.e. by colour change. The extent of enzymatic reaction is then a quantitative indication of the amount of antigen trapped by the primary antibody.
Elution
Removal by dissolving, such as the removal of adsorbed material from an adsorbent by means of solvents.
Enation
Laminar or cup-shaped outgrowth found on the underside of grapevine leaves effected by enation disease.
Enzyme-labelled antibody
See Conjugated molecule and ELISA.
Epinasty
An increase or decrease in growth of the upper or lower leaf surface or vein which causes the leaf to bend downward.
Ethidium bromide
An intercalating agent which allows the ready detection of double-stranded nucleic acid molecules in agarose gels. The nucleic acid/ ethidium bromide complex fluoresces brightly when exposed to ultraviolet (UV) light (ethidium bromide is highly carcinogenic).
Eye (of a bud)
The protruding meristematic portion of a bud which later enlarges and grows into a young shoot or flower.
Flecking
As in leaf flecking. Usually a lighter translucent spot or small patch on leaves.
Flush
The new, young and fresh growth of shoots and leaves.
Foundation tree
In a certification programme, the foundation tree is the primary tree derived from budwood that has been specially selected, shoot-tip grafted and/or heat-treated. It has been indexed and certified as virus-free and also true-to-type. It will become the primary source tree for all future progeny trees. A foundation tree can be synonymous with a mother tree or mother block tree.
Gel
The inert matrix used for electrophoretic separation of nucleic acids or proteins. Agarose gels are used for separation of DNA, agarose or polyacrylamide for RNA, and polyacrylamide for proteins.
Graft transmission
The transmission of a virus or other pathogen(s) by grafting tissue from the suspect host to an indicator plant.
Hybridization
The formation of stable duplexes between complementary nucleotide sequences via Watson-Crick base pairing. The efficiency of hybridization is a test of sequence similarity. DNA-DNA, DNA-RNA and RNA-RNA hybrids may be formed.
In classical genetics, and particularly plant breeding, hybridization means the formation of a novel diploid organism either by normal sexual processes or by protoplast fusion.
Immunoassay
An assay system in which a protein is detected using an antibody specific to that protein. A positive result is seen as a precipitate of an antibody-protein complex. The antibody can be linked to a radioactive atom (radio immunoassay) or to an enzyme that catalyses an easily monitored reaction (see ELISA).
Immunoblotting
A procedure whereby either the antigen or antibody molecules are bound to a proteinbinding substrate, such as cellulose nitrate, and then exposed to the complementary antigen or antibody. The antigen-antibody complex which forms on the membrane is detected by an appropriately labelled antibody.
Immunodiffusion
A procedure in which antibody and/or antigen molecules are allowed to migrate through an inert medium. A visible precipitate forms at the zone where related antigen and antibody molecules meet in a suitable concentration and react.
Immunofluorescence
The effect when antigens are detected, often within tissues, by use of an antibody to which a fluorescent material is attached.
Immunoglobulin
A blood serum protein that functions as an antibody, commonly a gammaglobulin.
Immunosorbent
A material that can adsorb serologically active molecules (antigens or antibodies). Cellulose nitrate and some plastics, such as certain polystyrenes, are good immunosorbents. Adsorbed molecules typically retain serological functions.
Inclusion bodies
Cytopathic intracellular structures found in virusinfected plants. They contain virus particles, other proteins or structures specific to the virus and/or formed as a result of virus infection.
Indexing
Any testing that will consistently confirm the presence (or absence) of a transmissible pathogen or identify a disease. The index test should be specific for the pathogen or disease.
Indicator plant
A plant used to test or index for the presence of a transmissible pathogen. The inoculated indicator plant will usually show very specific symptoms, thus permitting the diagnosis of a particular disease.
Infection feeding
After acquisition feeding where the pathogen is ingested by the insect, infection feeding is the secondary or follow-up feeding where the pathogen is injected into the host plant by the insect.
Inoculation
The process of infecting an indicator plant, usually by graft, mechanical or vector transmission.
Inoculum tissue
Tissue containing the transmissible pathogen or pathogens.
Intermediate antibody
An antibody used in the second step of an indirect ELISA assay. The intermediate antibody reacts to the antigen bound to the plate but is not labelled. It is detected by using another antibody which is labelled and is specific only for the intermediate antibody.
Linearization
The conversion of a nucleic acid that is normally circular into a linear form of the molecule, done by cutting the circular form at a single site.
Line pattern
Translucent, chlorotic or bright yellow lines, sometimes in a pattern resembling the outline of an oak leaf, induced in leaves of naturally infected vines or in indicators, mainly by nepoviruses.
Loading
As in loading ELISA plates. In ELISA, it is the process of adding a given amount of sample, buffer or any substance to the wells of an ELISA plate or gel apparatus.
Mealybugs
Scale insects of the family Pseudococcidae, vectors of some grapevine closteroviruses (see Leafroll).
Mechanical transmission
Transfer or transmission of graft-transmissible pathogens by means other than grafting and not involving vectors. This can be done by knife cut, razor slash, hand or cotton rubbing of sap on leaves using carborundum powder, or any other non-grafting method.
Molecular hybridization
See Hybridization
Molecular probe
See Probe
Mollicutes
One of the four divisions of the kingdom Prokaryotae, characterized by having no cell wall or peptidoglycan. This division includes the mycoplasmas and mycoplasma-like organisms (MLOs).
Monoclonal antibody
An antibody preparation containing only a single type of antibody molecule. Monoclonal antibodies are produced naturally by mycloma cells. A mycloma is a tumour of the immune system. A clone of cells producing any single antibody may be prepared by fusing normal lymphocyte cells with mycloma cells to produce a hybridoma.
Mother trees or mother block trees
Similar or synonymous with foundation or foundation block trees (see Foundation tree).
Negative stain
An electron-dense solution used to provide contrast around virus particles viewed on a transmission electron microscope.
Nematodes
Small, soil-inhabiting, root-feeding, worm-like animals, vectors of several grapevine nepoviruses.
Nick translation
A procedure to insert radioactive or other tagged bases in a DNA probe. It is a process whereby damaged dsDNA molecules are repaired with nucleotides, some of which are radioactive. It is a good way to repair a probe.
Nitrocellulose membrane (cellulose nitrate)
A nitrated derivative of cellulose which is made into membrane filters of defined porosity, e.g. 0.45 ?m, 0.22 ?m. These filters have a variety of uses in molecular biology, particularly in nucleic acid hybridization experiments. In the Southern and Northern blotting procedures, DNA and RNA, respectively, are transferred from an agarose gel to a nitrocellulose filter. Some centrifuge tubes are made of nitrocellulose; they are readily punctured with a hypodermic needle, and are frequently used for sucrose gradient centrifugation.
Northern blot, Northern transfer
A procedure analogous to Southern transfer, but in this case RNA, not DNA, is transferred or blotted from a gel to a suitable binding matrix such as a nitrocellulose sheet. Single-stranded RNA is separated according to size by electrophoresis through an agarose or polyacrylamide gel; the RNA is then blotted directly on to the support matrix with no denaturation. RNA fixed to the supporting matrix can then be hybridized with a radioactive singlestranded DNA or RNA probe.
Nucleic acid
A DNA or RNA molecule, which can be single or double stranded.
Nucleotide sequence
The order of alignment of nucleotides of nucleic acid molecules (see Sequencing).
PAGE
Polyacrylamide gel electrophoresis (see Polyacrylamide gels). A method for separating nucleic acid or protein molecules according to their molecular size. The molecules migrate through the inert gel matrix under the influence of an electric field. In the case of protein PAGE, detergents such as sodium dodecyl sulphate are often added to ensure that all molecules have uniform charge. Secondary structure can often lead to the anomalous migration of a molecule. It is common, therefore, to denature protein samples by boiling them prior to PAGE. In the case of nucleic acids, denaturing formamide, urea or methyl mercuric hydroxide is often incorporated into the gel itself, which may also be run at a high temperature. PAGE is used to separate the products of DNA-sequencing reactions, and the gels employed are highly denaturing since molecules differing in size by a single nucleotide must be resolved.
Pegs
Needle-like growths or pinpoint projections observed on wood or bark, which may be symptomatic for certain diseases of grapevine. Pegs usually have corresponding pits on the opposite bark or wood surface.
Peptidoglycan
Polysaccharide chains covalently cross-linked by peptide chains. The presence or absence of peptidoglycan in the cell walls of bacteria is used to distinguish gracilicute-like organisms (which contain peptidoglycans) from mycoplasma-like organisms (which have no peptidoglycans).
Plant laboratory
A sophisticated greenhouse designed and used primarily for indexing.
Plate-trapped antigen
Antigen adsorbed directly on the ELISA plate without use of a trapping antibody. For example, virus particles can be trapped to the surface of ELISA plates from extracts of infected tissue added to the wells of the plate. Other proteins, however, are also adsorbed.
Polyacrylamide gels
Often referred to, incorrectly, as acrylamide gels. Gels made by cross-linking acrylamide with N, N'-methylene-bis-acrylamide, used for the electrophoretic separation of proteins and also RNA molecules. DNA is usually too heavy to migrate far in polyacrylamide. Polyacrylamide beads are also used as molecular sieves in gel chromatography and are marketed under the brand name Nio-gel.
Polyclonal antiserum
Antiserum harvested from the blood of immunized animals. Polyclonal antisera contain a mixture of antibodies to the various antigenic molecules present in the material used to immunize the animal. In contrast, monoclonal antisera contain only a single antibody.
Potting mix
A mixture of ingredients used as an artificial soil medium for container growth of plants.
Primary leaves
The first emerging leaves from a germinating seed. These leaves may be the cotyledons or may differ in shape from the secondary leaves.
Primer
A low-molecular-weight species that promotes a reaction, such as an oligonucleotide that binds to a template, permitting a copy of the template to be further synthesized.
Probe
Noun: a specific DNA or RNA sequence that has been radioactively labelled to a high specific activity. Probes are used to detect complementary sequences by hybridization techniques such as Southern or Northern blotting or colony hybridization.
Verb: to hybridize in order to detect a specific gene or transcript, e.g. "We probed our bank with labelled viral RNA to detect clones containing viral DNA sequences".
Probe denaturation
Treating the probe under conditions that will separate its nucleic acid strands and permit their subsequent hybridization with the target molecules to be tested.
Probe labelling
Investing probes with detectable tags by a variety of procedures.
Prokaryotes
Bacteria-like organisms in the kingdom Prokaryotae that have no organized nucleus and are surrounded by a nuclear membrane.
Protein A
A protein with a high affinity for antibody gammaglobulins.
Radioactive probe
A nucleic acid that has been made radioactive by one of several techniques (e.g. nick translation) and is to be used to detect a complementary nucleic acid sequence.
Recombinant plasmid
A bacterial plasmid DNA containing an insert of DNA from a non-related source, e.g. a plasmid containing an insert of viral cDNA. It is created by recombinant DNA technology.
Replicative form (Rf)
The intracellular form of viral nucleic acid which is active in replication. For example, M 13 phage particles contain a single-stranded DNA circle, while the Rf of the same molecule is double stranded.
Restricted endonuclease
An enzyme that recognizes and cuts doublestranded DNA at specific sites determined by the sequence of bases at that site.
Resuspension medium (RM)
See TKM buffer
Reverse transcription enzyme
The enzyme that accomplishes the enzyme synthesis of a copy DNA from an RNA template in the presence of a primer and nucleotide triphosphates under appropriate conditions.
Ringspot
Circular, ring-like translucent, chlorotic or bright yellow spots induced in leaves of naturally infected vines or indicators, mainly by nepoviruses.
RNA
Ribonucleic acid. The alternative reservoir of genetic information besides DNA. Viruses have single-stranded or double-stranded RNA genomes. In organisms, RNA is transcribed from DNA and is essential for the expression of the genetic information contained within the DNA. RNA differs from DNA in having ribose instead of deoxyribose as the sugar moiety in its nucleotides and in having uracil instead of thymine as one of its two pyrimidine bases. RNA, but not DNA, may be degraded by al kaline hydrolysis.
Rugose
As in rugose wood, meaning rough or wrinkled, with pits and grooves on the woody cylinder.
Secondary leaves
In contrast to the primary leaves of a germinating seed, the secondary leaves are permanent leaves with a fixed morphology. Many seedlings produce both primary and secondary leaves, but many have just one type.
Sequencing
The determination of the order of nucleotides in a DNA or RNA molecule, or that of amino acids in a polypeptide chain.
Sequencing gel
A long polyacrylamide slab gel that has sufficient resolving power to separate singlestranded fragments of DNA or RNA that differ in length by only a single nucleotide.
Electrophoresis is carried out at high voltage and with the gel in a vertical position. Urea is usually included in the gel mixture as a denaturing agent. This prevents internal base pairing within the single-stranded molecules and ensures that their relative speed of migration is solely dependent on their length.
Shoot-hp grafting
A micro-grafting procedure. The meristematic growing tip (the meristem plus one to three leaf primordia) is excised by cutting the very young tip using the cutting edge of a razor-blade mounted in a special handle. This decapitated tip is then grafted to a very young seedling with the aid of a binocular microscope. The grafted plant is usually then grown in vitro and later transplanted or grafted to produce a shoot-tip grafted plant or tree.
Shot berry
A disorder in which grape bunches bear small, imperfectly developed berries. It is induced and/ or enhanced by viral infections (nepoviruses in particular).
Slot-blot
See Dot-blot
Southern blot, Southern transfer
A technique that combines the resolving power of agarose gel electrophoresis with the sensitivity of nucleic acid hybridization. DNA fragments separated in an agarose gel are denatured in situ and then blotted or transferred, usually by capillary action, from the gel to a nitrocellulose sheet or other binding matrix placed directly on top of the gel. Single-stranded DNA binds to the nitrocellulose and is then available for hybridization with labelled, 32P or biotinylated, single-stranded DNA or RNA. The labelled nucleic acid is known as the probe and, in the case of DNA, is often prepared by nick translation. The hybrids are detected by autoradiography, in the case Of 32p, or by colour change, in the case of a biotinylated probe. A very sensitive and powerful technique, it is often described as "blotting".
STE buffer
Sodium chloride, Tris and EDTA (see Part 111). Used in purification of nucleic acids including dsRNA and viroids.
Stem pitting
Alteration of the xylem consisting of groovelike or pit-like depressions of the woody cylinder, as in rugose wood.
Streptavidin
A microbial protein which binds biotin. It is preferred to avidin because of its more specific binding ( see Biotin). In molecular hybridization, streptavidin reacts specifically with the biotin molecules fixed on the probe.
Stub
As in a rootstock stub. The short projecting portion of the stem that remains after the rootstock is severed from the growing scion. The stub is that small portion above the scion which should be trimmed flush with the scion at a later time.
Substrate
The substance acted upon by an enzyme. Usually this substance contains a chemical which, when acted upon by an enzyme, will undergo a colour change that can be easily seen and measured.
Symptomless carrier
A tree or plant that contains a grafttransmissible pathogen but shows no symptoms. An example of a symptomless carrier would be an American rootstock containing leafrollassociated closterovirus.
Synergism
A phenomenon in which the joint action of more than one agent such as two viruses in combination induces a more intense symptom in an indicator plant than the agents could induce separately.
TAE buffer
Tris, sodium acetate 3H2O and sodium EDTA.
Template
The molecule that is acted upon or copied, as in the production of cDNA probes by reverse transcriptase.
Template primer
See Primer and Template
Thermotherapy
Treatment of budwood or plants by heat to eliminate internal pathogens.
TKM buffer
A buffer with Tris, KCI and MgCl2
TME
As in TME Tris buffer. Tris, MgCI2 and EDTA
Trapping antibodies
Antibodies used to coat the wells of ELISA plates (the first layer in sandwich assay procedures). Antibodies adsorbed to the solid surface of the plate trap related antigens from the sample extracts placed in the plate for testing.
Triturating
Rubbing or grinding, as with tissue ground with pestle and mortar.
Viroid
A small molecular RNA, transmissible in plants, which has no extracellular protein component or translation capacity and which can be pathogenic. It is composed of naked, singlestranded, low-molecular-weight RNA (MW80000 to 130 000) which utilizes only host components for its replication. Viroids exist in solution as rod-like structures arranged in a series of short basepaired and non-base-paired regions.
Virus
Viruses are macromolecular transmissible agents capable of causing diseases in plants and animals. They are small enough to pass through a millipore filter of 0.2 ?m. They have been considered to be either living organisms or simply a molecular complex of nucleic acids and proteins capable of multiplication in living cells. Viruses are characterized by a core of nucleic acids with a genome of less than 3 x l 08 daltons surrounded by a protein coat that can induce formation of antibodies.
Western blot
A procedure used to determine the presence of viral coat protein in plant tissue extracts. Extracts are electrophoresed in polyacrylamide gel slabs; protein bands are then electrotransferred from slabs to an appropriate support matrix (e.g. PVDF membrane) and exposed to specific antiserum for recognition.
Yellows
Disease induced by mycoplasma-like organisms (MLOs), characterized by rolling of leaves, generalized or sectorial yellowing or reddening of the leaf blade, irregular ripening of canes and withering of bunches.