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[email protected] • Website: http://cost.cordis.lu R.P.M.861.794.916 – Tribunal de Commerce de Bruxelles Short term Training Mission – Plant Bacteriology MANUAL 3rd – 7th March 2008 Central Science Laboratory York, UK This manual was compiled by: John Elphinstone David Stead Neil Boonham Jenny Tomlinson Richard Thwaites Neil Parkinson Helena Stanford Elspeth Steel 2 Table of Contents: 1. Bacterial diseases of nuts and stone fruits ......................................................... 4 1.1 Symptoms .................................................................................................... 4 1.2 Isolation and identification of bacterial pathogens of nuts and stone fruits.. 16 1.3. Confirmatory diagnosis.............................................................................. 18 2. Bacterial Taxonomy and Nomenclature ........................................................... 21 2.1 Classification .............................................................................................. 21 2.2 Identification ............................................................................................... 21 2.3 Nomenclature............................................................................................. 21 2.4 Diagnosis ................................................................................................... 21 2.5 Gram Positive Bacteria............................................................................... 22 2.6 Gram Negative Bacteria, within the alpha Proteobacteria........................... 23 3. Primer design and Bioinformatics .................................................................... 24 3.1 Database searching ................................................................................... 24 3.2 Retrieving sequence information ................................................................ 25 3.3 BLAST searching ....................................................................................... 26 3.4 Making a multiple sequence alignment....................................................... 29 3.5 Primer design theory .................................................................................. 31 3.6 Primer design practice................................................................................ 32 4. Isolation of bacterial DNA ................................................................................ 33 4.1 DNA isolation using ChargeSwitch Technology (CST) ............................... 33 5. PCR and real-time PCR protocols ................................................................... 35 5.1 Conventional PCR...................................................................................... 35 5.2 Real time PCR ........................................................................................... 35 5.3 rep-PCR ..................................................................................................... 37 5.4 Protocols .................................................................................................... 38 6. Minimum requirements for diagnosis ............................................................... 45 6.1 Diagnosis or Identification: Detection or Diagnosis ..................................... 45 7. Fatty acid profiling............................................................................................ 48 7.1 Summary.................................................................................................... 48 7.2 Introduction ................................................................................................ 48 7.3 FAMEs ....................................................................................................... 49 7.4 Methods ..................................................................................................... 50 8. Identification of bacteria by partial gene sequencing........................................ 52 8.1 Introduction ................................................................................................ 52 8.2 Procedures................................................................................................. 52 9. Immunofluorescence cell staining for detection of bacterial pathogens ........... 57 9.1 Introduction ................................................................................................ 57 9.2 Material and equipment .............................................................................. 57 9.3 Method ....................................................................................................... 57 10. EUPHRESCO – ERA-NET Leaflet ................................................................. 59 3 1. Bacterial diseases of nuts and stone fruits David E. Stead Central Science Laboratory, Sand Hutton, York, YO41 1LZ, UK
[email protected] 1.1 Symptoms 1.1.1 Bacterial canker, spot and shothole of plum and peach (Xanthomonas arboricola pv. pruni) Plum, peach, almond cherry and apricot are susceptible. Initial symptoms are typically angular leaf spots with chlorotic margins. Initially, small chlorotic lesions occur on leaves. On the lower surfaces these have tan centres, becoming visible from the upper surfaces as they enlarge, and then becoming darker brown maroon or black. Lesions are often surrounded by a chlorotic halo and tend to be more numerous at the shoot tips. Ooze may develop from them. Necrotic areas often drop out, leaving shotholes. On peach stems, spring cankers often enlarge to cause ‘black tip’ as dark-green watersoaked blisters on the tips of overwintering twigs. These enlarge and kill the growing shoots. Summer cankers arise as watersoaked purplish, sunken lesions around infected lenticels, later becoming dark and sunken. On twigs, deep-seated, perennial cankers occur. Infected stems may become deformed and die. On plum and apricot stems, perennial cankers form, the inner bark is discoloured and dieback is common on these hosts. Lesions on fruit of all hosts are similar – small, circular, dark-brown and pitted. On peach they are often surrounded by a pale-green halo. As fruits enlarge the lesions crack and may exude yellow, bacteria-laden ooze. A B 4 C D E F G 5 The disease is characterised by black lesions. University of Tuscia). Jones. Balestra. Jones. University of Tuscia). H) symptoms on cherry fruit (G. M. Jones. CABI). Bacterial blight of walnut (Xanthomonas arboricola pv. CABI). Jones. CABI). C) Cankers on peach twigs at bud break in spring (Alan L. CABI). A B 6 . B) Spotting on peach fruit (Alan L. Jones. widespread and sometimes serious disease in most walnut producing areas. 1. CABI). F) Symptoms on Stanley plum fruit (Alan L. juglandis) Bacterial blight of walnut is a common. Jones.1. long and narrow on young stems and irregular often large on fruits.H Symptoms caused by Xanthomonas arboricola pv pruni: A) Yellowing and tip burning on peach leaves (Alan L. G) Pitting and gumming on nectarine fruit (Alan L. D) Spotting on nectarine fruit (Alan L. CABI). 2. angular to irregular on leaves. E) Cankers and gumming on cherry tree (G M Balestra. 7 . E) Transverse section of fruit showing necrosis. INRA Symptoms of walnut blight: A) Canker on walnut twig.C D E Pictures L Gardan. C) Spots on new fruit. B) Brown necrosis on green twig. D) Sunken spots on new fruit. Image from S Sule Leaf spot on walnut 8 . corylina). Black spots and streaks may be found on young stems and cankers may also be found on twigs and branches. Infected leaves show small. Young green nuts may also show small. necrotic spots. Bacterial blight of filbert and hazel (Xanthomonas arboricola pv. black. A B C D 9 .1.3. brown or black spots.1. angular to irregular. One of the most characteristic symptoms is necrosis of the emerging buds in late spring. syringae and P. Spur leaves tend to be resistant as soon as 10 . are formed in the autumn or winter. peach almond. which are not perennial. In the UK and most of north-west Europe the morsprunorum pathovar is the usual cause of disease but the syringae pathovar is also important in many other fruit-growing areas of Europe.E F Images coutesy of L Gardan. shot hole and bacterial canker of cherry and plum (Pseudomonas syringae pv. Once blossom fall occurs the progress of cankers is arrested and populations of bacteria within the cankers decline and usually die out. INRA Symptoms of bacterial blight on hazel: A) Bud destruction and dieback. D) Canker with cracks on bud. syringae pv. apricot and many other Prunus spp. 4 Leaf spot. but do not increase much in size until the following spring. The disease caused by the morsprunorum pathovar has a well defined seasonal cycle with a winter canker phase alternating with a summer leaf disease. plum. The disease is most common on cherry and plum in Europe. There are strains specific for each host.1. Cherry strains are cultivar specific and at least 2 races are recognized. morsprunorum) As well as causing cankers on the stems and branches of stone fruit trees (cherry. F) necrosis with halo on young hazel nut. when they enlarge rapidly. E) Close up of angular leaf spots. At the same time the leaf infection phase occurs. Cankers. killing large areas of green bark. shoots and fruits. B) Yellowing on young leaves. C) Leaf spot symptoms on hazel leaf. 1.) the bacteria also infect leaves. linear depressions in the bark. trees of all ages are susceptible and most cankers are found at sites of leaf scars on fruiting spurs. morsprunorum bacteria gain access to woody tissue only via wounds in leaf scars and bark. death ensues. Unlike P. which may drop out to give a shot hole effect. Cankers can also be located on the branches. On younger thin-barked branches cankers are first visible in the spring as shallow discoloured sunken bark lesions often showing the presence of amber-coloured. gummy exudates. In plum.they mature. This usually results in die-back of the spur but may occasionally spread to form a canker in the parent branch. Gumming is less common and not so obvious as on cherry. often coalescing to form large irregular necrotic spots. syringae pv. The disease caused by the syringae pathovar has a somewhat different epidemiology. Symptoms vary between host species. Spots on leaves are caused by both pathovars and are usually reddish-dark brown. In cherry. Rapid multiplication occurs in the spring. However. especially on the crotch and angles between the branches. cankers occur most frequently on stems and trunks often leading to death of mature trees. A B 11 . but young leaves on extension shoots are infected. rounded or angular. Cankers can be perennial and the bacteria overwinter in them as small populations. ooze is often produced and the bacteria spread to leaves by rain splash. Once the stem is girdled. from which new cankers arise. bacteria multiply as epiphytes on all leaves during the summer and reside until leaf fall. being the main source of new canker infections via the exposed leaf scars. When a canker girdles a branch dieback will occur. Cankers may extend the length of the stem and often appear as dark-coloured. 12 . CABI). Jones. CABI). Jones. CABI). CABI). Jones. CABI). C) Shothole symptoms on plum (AgrEvo. D) Symptoms on cherry fruit (Alan L. Jones. Canker and gummosis on sweet cherry branch (Alan L. A) leaf spot and shotholes on sour cherry (Alan L.C D E Symptoms of Pseudomonas syringae on cherry and plum. B) Spur dieback and necrosis of mid-vein on Stanley plum (Alan L. A B 13 . On fruits. persicae). In spring. especially in nectarine. olive to dark brown. Initially small.tracing down to larger branches reveals canker developments. These are brown and dry. In autumn and spring. In spring.5 Bacterial decline and canker on nectarine and peach (Pseudomonas syringae pv. water-soaked lesions appear initially. This symptom is negligible in peach. The pathogen may be isolated from these. On main leaders and trunks.1. The pathogen can usually be isolated. It is difficult to isolate the pathogen from these lesions. investigation of dark discoloration of bark on previous year's growth reveals extensive brown and water-soaked lesions in the cambium. deep incisions into wood of apparently healthy stems reveals discoloured wood . olive. small. water-soaked lesions appear on leaves giving rise to necrotic spots of 1-2 mm diameter. Necrotic tissue falls out giving rise to 'shot-holes'. small 2-5 mm round to elliptical lesions. In favourable conditions. olive. the pathogen can also be isolated from root tissues below cankers. These can be associated with the exudation of gum. occur on shoots at nodes and on internodes. Alternatively.1. There is no visible sign of these lesions which are only detected when withering of leaves signals the girdling of the branch. This is a disease primarily of peach and nectarine but myrobalan and Japanese plum may also be affected. Infected root tissue shows no sign of invasion. extensive brown lesions are found with no discrete margin to healthy tissue. these spots continue to expand during the spring and can cause severe distortion to developing fruit. cherry. 14 .C Images courtesy of Landcare Research New Zealand Symptoms of bacterial decline and canker on nectarine. galls become more woody and fissured with age. A) symptoms on nectarine trunk. rounded. usually just above soil level.6 Crown gall of almond.1. Roots have rounded galls similar to those on stems but usually much smaller. smooth or fissured galls. plum and walnut (Agrobacterium tumefaciens). 1. pecan. hazel. Crown gall is a common disease affecting a very wide range of host plants. peach. On perennial woody hosts. although occasionally galls are formed on petioles. apricot. On most hosts leaves are unaffected. Stems have initially neat. C) Infection and distortion of nectarine fruit. can be more than 10 cm across and can girdle the stem. B) Necrotic bark and cortex of nectarine branch. W. Moore. Moore.Crown gall on walnut (Image: Larry. courtesy of CABI) 15 . W. courtesy of CABI) Crown gall on flowering cherry (Image: Larry. 5% sucrose nutrient agar The growth on these isolation plates may enable a presumptive diagnosis to be made See Table 1. and even then their numbers may be too low to detect readily. Streak the suspension on plates of nutrient dextrose agar. The pathogen may only be present in small numbers or in reasonable numbers only in small pockets of the tissue of dormant cankers. rough or contaminated with soil. Slice pieces of young. King’s medium B plate d. Lift the epidermis with the pint of the scalpel and with blunt nosed forceps.1. Cut into the lesion with a scalpel to determine the position of its margin and then make an incision in the epidermis of the healthy tissue parallel to and a millimetre or so from the margin.5% available chlorine) for 10 minutes and rinse thoroughly in 3 changes of sterile water. surface sterilize by soaking in hypochlorite solution (0. tear it back to expose the leading edge of the lesion. Wash the gall in tap water.g. It is often worthwhile sampling from a wide variety of samples and perhaps pooling them to facilitate isolation. 5. immerse in a suitable wetting agent (e. If bacteria cannot be detected immediately. If the gall is old. 3.1% Manoxol). Nutrient dextrose agar plate c. 6. 3. fresh tissue from the gall surface. Leave to stand for at least 30 minutes. 16 . Examine microscopically for Gram negative. Crush in a few drops of sterile water in a sterile Petri dish. Leave the crushed gall for at least 20 minutes and preferably for several hours to allow the bacteria to diffuse out of the tissues. 2. Dissect out part of the edge with a flamed scalpel and tease it out in sterile water. short. 4. Streak suspensions containing bacteria on the following media: a. 4.2 Isolation and identification of bacterial pathogens of nuts and stone fruits 1. 2.2. clean it thoroughly by scrubbing gently.1 Isolation of Xanthomonas and Pseudomonas from cankers 1. 0. Isolation of Agrobacterium tumefaciens from galls 1. Nutrient agar plate b. it is often necessary to examine a number of the lesions microscopically to find these pockets. rod-shaped bacteria. Wash cankers in running water. although this is also likely to increase the number on non-pathogenic bacterial colonies on the isolation plates. creamy to yellow. whitishgreen to yellowish-brown. smooth. domed. and are the rule in P. smooth. yellowish-orange Coryneform 1 2 3 Occasionally atypical. smooth. smooth or wrinkled. High convex to domed smooth. diffusible or non-diffusible pigments may be produced Non-fluorescent pseudomonads High convex to domed. syringae pv. s. mucoid. No diffusible pigment. domed. pv. IVa Whitish-grey. mucoid. raised As above As above Green fluorescent pseudomonads of group Ib. smooth. smooth. convex or domed. Erwinia amylovora produces whitish. smooth mucoid (occasional levan 2 production ) usually white to whitishgrey Raised or convex. raised with diffusible yellowish green pigment that fluoresces blue-green under 1 ultraviolet light Whitish-grey. creamy to yellowish As 5% SNA. morsprunorum. mucoid (levan). persicae. Gram positive. brown diffusible pigment produced rarely. Pink. domed. convex to domed. Whitish-grey. whitish to yellowish with greenish centre As above As 5% SNA Green fluorescent pseudomonads of group II Convex. Xanthomonads Raised. non-fluorescent forms of some pathogens of Group I are isolated. mucoid levan colonies. blue or violet pigments are sometimes produced 17 . butyrous Green fluorescent pseudomonads of group Ia. yellowish to brown. yellowish-orange Raised or convex.Table 1: Characteristics associated with colonies of pathogens on isolation plates 5% sucrose nutrient agar King’s B Nutrient dextrose agar Possible pathogen Whitish. whitish-grey Soft rot erwinias Convex. but they are common in P. whitish green. IVb Convex. diffusible or non-diffusible pigments may be produced Whitish-grey but often producing pigments which do not fluoresce under ultraviolet light Convex. smooth white3 yellowish Raised. mucoid. III. raised. whitish-grey which do not fluoresce under ultraviolet light Raised or convex. smooth or wrinkled. Inhibited by 0. syringae pv. P. colonies of agrobacteria on all media should be neat. Oxidase reaction negative 3. morsprunorum isolates do not always produce this pigment. creamy to yellowish in colour on all media and with no diffusible pigment produced on King’s B agar. syringae pv syringae isolates produce a diffusible yellowish-green diffusible pigment on King’s B medium which fluoresces blue green under ultraviolet light. 3-keto lactose produced on lactose yeast extract plates after 2 days by biovar I strains but not by biovars II or III. Xanthomonas colonies isolated from affected tissue with typical disease symptoms should be high convex to domed.3.3 Agrobacterium After 2-3 days incubation at 25 ºC. entire. Further characteristics of Xanthomonas include: 1. domed. smooth. Additional properties of agrobacteria include: 1. Pseudomonas syringae colonies isolated from affected tissue with typical disease symptoms should be whitish. syringae pathovars syringe and morsprunorum are shown in Table 2. Tobacco hypersensitivity positive Characteristics differentiating P. morsprunorum pv. Table 2: Distinguishing characters between Pseudomonas syringae pathovars morsprunorum and syringae. Levan positive 2.1% tri-phenyl tetrazolium chloride (TTC) 1. Oxidase reaction negative 3. All agrobacteria are O/F test positive (oxidative) 18 . smooth.2 Pseudomonas After 2-3 days incubation at 25 ºC. domed and mucoid.3. Confirmatory diagnosis 1. syringae 5% sucrose nutrient broth White growth Yellow growth Recovery from 5% nutrient agar after 6 days - + Aesculin or arbutin hydrolysis - + Gelatin liquefaction - + Brown diffusible pigment on King’s medium B +/- - Green-fluorescent diffusible pigment on King’s medium B +/- + 1. Gram stain negative 2. Character pv. Both pathovars have the following properties common to LOPAT group Ia psedomonads: 1. P. Catalase reaction positive 4.1.1 Xanthomonas After 3 days incubation at 25 ºC. Pectate liquefaction negative 4. 2. Arginine dehydrogenase negative 5. round.3.3. smooth and mucoid (levan positive) on SNA medium. Where possible grow bacteria on nutrient agar.4. Tobacco plants should be held at 16 ºC for 4 days prior to inoculation and should be incubated at 33 ºC after inoculation. Use large-leaved rapidly grown tobacco plants. 4. For Pseudomonas spp. 19 . Not all xanthomonads give a positive hypersensitive reaction in tobacco.3-0. 7. If growth cabinets are available incubate at 25 ºC and 85% relative humidity with a diurnal daylight regime of 16 hours. Growth on selective media can be used to indicate biovars (see Table 3) Table 3: Isolation of Agrobacterium tumefaciens biovars on selective media Medium Selective for biovar Colony colour Schroth I Yellow/tan Kerr II Pearly white/tan Roy and Sasser III Pinkish-white (red centre) Hypersensitive reaction (HR) tests 1. Stab soft stems of plants with a needle charged with culture (106-107 cfu per ml). Inject the mesophyll of the leaf lamina with the suspension by inserting the needle of a hypodermic syringe into the cavity which runs along the side of the lateral veins. but at least within 48 hours. 1. 3. incubate plants in a well-ventilated glasshouse at a temperature of less than 30 ºC. A positive hypersensitive reaction is given by a rapid collapse and watersoaking of inoculated tissue. light brown necrosis of the watersoaked tissue within 3 days.6 mm external diameter. It may be possible to produce spreading lesions and dieback on young detached shoots standing in water. Inject sufficient inoculum to flood the intercellular spaces of the mesophyll – apparent by watersoaking of the tissue – but not at such high pressure that blisters appear on the leaf surface. Yellowing or browning without collapse is not a positive reaction. It is therefore recommended to also use tomato or pepper seedlings. The diagonal of the needle aperture should be adjacent and parallel to the surface of the tissue. The reaction is generally weaker than for fluorescent pseudomonads. Under ideal conditions. Adjust an aqueous suspension from a 24-48 hour culture to a cell density equivalent to 108-109 cfu per ml (optical density of approximately 0. usually within 24 hours. followed by a dry.4 at λ = 600 nm). Inject more than one leaf and more than one sector per leaf for each organism. Arrange test and control injections on opposite sides of the main vein. 5. Label the areas of the leaf injected. It is necessary to use a narrow gage needle of approximately 0. 2. Host test for canker development Perform a host test using healthy susceptible species: 1.3. 6. 2. 3. Inject a separate area of the leaf lamina with sterile distilled water as a control and with a known reference culture of the suspected pathogen (known to induce the HR) as a positive control. White Burley is a commonlyused cultivar. Symptoms appear more reliably and quickly if the site of inoculation is protected from desiccation by covering with polythene for 48 hours. a positive result should be obtained within 24 hours. Spray inoculum as a fine mist until the plant surface is wet. fan-shaped spray. juglandis as a positive control. to prevent drying out. 3. 1. Ensure that the polythene does not touch the plant. Host test for Agrobacterium tumefaciens 1. Repeat tests using different methods and conditions may be necessary to ensure accurate diagnosis. If this is impractical. 5. Galls usually develop within 2 weeks. Any low pressure. Prepare a suitable inoculum by washing the growth from a nutrient agar or other suitable agar medium in sterile water. Tomato.7. 1. 7. Lesions are often elongated along stems. Lenticular cankers may be produced by incubating young shoots of plants in conditions of high relative humidity in a damp chamber or with a polythene bag tied over the stems before spraying with a suitable suspension. These should become necrotic. ensuring that upper and lower leaf surfaces are wetted. easily directed. use several hosts. The canker phase of some diseases cannot be produced by inoculation at all times of the year. 8. Mix well and dilute in sterile water to obtain a population of approximately 105-106 cfu per ml. wound infection usually succeeds in producing symptoms but caution is then needed in interpreting the results. 6. Use a known reference culture of Xanthomonas arboricola pv. Grow the plant on at approximately 20-25 ºC and 85% relative humidity with 12-16 hours light. is very convenient and produces a fine. Prick one or two labelled leaves on each plant and the upper part of a young stem or young petiole in about 5-6 places with a sterile needle. 3. 5. the tip of which has been bent at right angles to the bevel by pressing it hard on a surface. Host tests for leaf and stem spot symptom development 1. Incubate at 25 ºC in closed boxes lined with damp blotting paper. 4.4. 2. 2. rounded or angular. Host test on immature walnut fruits for Xanthomonas arboricola pv. Swab immature healthy fruits with alcohol and wash in sterile water. easily sterilizable sprayer can be used. A hypodermic syringe with a needle. Place a drop of inoculum (106 cfu per ml) on the fruit surface and puncture the fruit by pricking through the drop with a sterile needle.6. Use sterile water as a negative control. 20 . sunflower. 4. and cover immediately. juglandis. 1. Kalanchoe sp. 3. Inoculate young plants covered with polythene sleeves 24 hours prior to inoculation.5. 5. If lenticular or stomatal infections do not occur. 2. usually brown-black and usually with a watersoaked margin. 1. The use of a positive control is essential for determining that he conditions of inoculation have the potential to produce infection and symptom expression following inoculation with a known reference strain. 4. Observe from 3 days onwards for typical watersoaked lesions. Observe for gall symptoms for up to 28 days. marigold. or place in a humid chamber. Use the original host where this is possible. Inoculate either by stabbing young stems or leaves with a needle through a droplet of bacterial suspension (containing approximately 108 cfu per ml). Incubate for 48 hours before removing the polythene sleeve or removing the plant from the humid chamber. and chrysanthemum are useful and easily cultivated. including the 3 pillars of taxonomy. clinical microbiologists are interested in the serotype and antimicrobial resistance patterns.2.2. Bacterial Taxonomy and Nomenclature David E. or to isolate and identify the organism that causes a disease. Although identification is an integral part of diagnosis. Between them they cover identification and diagnosis. The species is the gold standard here and is usually defined as a group of strains which share at least 70% DNA homology. pathovar. classification. other house keeping genes have been used. Most taxonomists prefer a polyphasic classification based on a number of different attributes. genus and species. identification and nomenclature. named and communicated. 2.4 Diagnosis Diagnosis is the process of determining the cause of a disease. often in groups of up to seven. biovar and race. there is a trend towards phylogenetic classifications based on sequences of genes that mirror the evolution of the organisms.3 Nomenclature Nomenclature is the means by which the characteristics of a species or other taxon are defined.gov. York. Unfortunately. Stead Central Science Laboratory. family. 21 . 2. easy to use. Sand Hutton.2 Identification Identification is the practical use of classification criteria to distinguish organisms from others. 2. polyphasic and phylogenetic classifications do not necessarily coincide. Initially this was largely based on the 16S rDNA but recently. Perhaps because these are time consuming and expensive. For example. fatty acid profiling is a useful method that broadly differentiates bacteria at species level. although two are nomenclaturally out of date (1. Current approved names are regularly updated (4). whereas plant bacteriologists are concerned with pathovars. YO41 1LZ. Historically this has proved contentious for a number of published names. generic methods. The classification results in clusters of strains defined at various taxonomic levels including class.stead@csl. although the approaches are somewhat different. There is nothing inherently scientific about classification and different groups of scientists may classify the same or similar organisms differently.3).1 Classification Classification is the orderly arrangement of bacteria into groups. UK d. a diagnosis can usually be made with fewer tests than an identification of the isolate. There are 3 manuals that are invaluable to diagnosticians (1. host specificity and virulence genes borne on mobilisable plasmids. There is an internationally accepted code for nomenclature. Within plant pathogenic species there may be further grouping at subspecies. 2). For example. in a process referred to as multi locus sequence typing (MLST). But there is a difference in an identification of a pure culture based on a fatty acid profile and a diagnosis of a disease based on a fatty acid profile of an isolate supported by typical symptoms. to verify the authenticity of a strain. Methods that can be used in identification include tried and tested traditional methods as well as modern molecular methods. Both processes have merit in the search for cost effective. 2. mostly through the upgrading and downgrading of taxonomic rank from and to pathovars and vice versa.uk This section reviews the current taxonomy of the plant pathogenic bacteria. Another problematic area is the identification of scab forming species within Streptomyces. it will be a long time before such methods are available to everyone and the more traditional methods must not be forgotten.g. However. This is best exemplified in the Enterobacteriaceae. This method could then be used in identification. where the genus Erwinia has been divided on the basis of phylogenetic differences within the 16SrDNA genes sequences.The addition of a host test raises the level from a presumptive diagnosis to a confirmed diagnosis. Curtobacterium. Genetic fingerprints. Identification at species level can thus be difficult and beyond the means of most diagnostic laboratories. The former may be suited to a grower. The first four of these are in the family Microbacteriaceae and are differentiated on the basis of 16S rDNA analysis. vesicatoria. The ideal is a classification based on a polyphasic approach for which a single generic method gives a parallel classification. most frequently differentiate these. euvesicatoria and X. nutritional and protein profiles are very useful in deciding whether strains are the same or not.e. As sequencing of DNA and whole genomes becomes more accessible. and Rhodococcus. groups of strains which have less than 70% DNA homology but for which there are few tests to differentiate them. There is no doubt that taxonomy should serve the needs of users such as diagnosticians and it is inevitable in the pursuit of more cost effective methods that identification and diagnosis will move towards more generic methods.g. bacterial spot of tomato and pepper with fairly similar symptoms can be caused by pathovars within several Xanthomonas species proposed as X. There are several fairly recent key taxonomic revisions that hinder identification and sometimes diagnosis: • Some diseases e. the latter to a finding of statutory significance. all in the Actinomycetales are Clavibacter. e. all of which are also useful in diagnosis. gardneri. genetic fingerprints and serological assays. then phylogenetic methods such as sequencing of housekeeping genes is likely to become a more popular and available method. Leifsonia. PCR. And whereas 16S data cannot always reliably differentiate species. • A fairly recent complication has been the concept of genomospecies. It is likely that gene sequencing will help here. sequencing of other housekeeping genes usually can. most of these are not named but within what was Xanthomonas campestris. . Streptomyces. • Another complication has been the mix of phenotytpic and phylogenetic classifications at different taxonomic levels. 2. Clavibacter and Curtobacterium both have single pathogenic species comprising subspecies and pathovars respectively. axonopodis pv. Some of the more recent evidence supports the use of 16S rDNA sequencing to differentiate genera. within the Enterobacteriaceae. Fatty acid profiles give other information of taxonomic value eg chemotaxonomic information at genus level. some 20 plus species have been classified and named. X. Rathayibacter. 22 . vesicatoria. Within Pseudomonas syringae. i.5 Gram Positive Bacteria The main genera. whereas classification of species within them is still based largely on a polyphasic approach. X. CABI. which shows good correlation with DNA homology. Again fatty acid analysis and genus specific PCR primers are useful for genus determination. J. fatty acids are useful in genus determination.3rd edition APS. Serratia). R. Diagnosis can be straightforward using traditional methods (2).E. A recent proposal subsumes it within Rhizobium but there is resistance to this. Species and many pathovars within them can be differentiated by repetitive sequence PCR as well as traditional and host tests (1. Traditional tests are still useful for diagnosis (1. Pseudomonas comprises many different species including genomospecies still classified within P.M. (1987) Methods for the diagnosis of bacterial diseases of plants. 3) and by fatty acid analysis and repetitive sequence PCR. Identification and diagnosis at species level is straightforward and there are species-specific PCR primers as well as a range of other tests (1. race and phylotype exist. Pectobacterium. Dickeya. Young. 2. Schaad. For Acidovorax. fatty acid analysis is also useful for genus determination. Burkholderia and Ralstonia. Burkholderia is a complex genus for which the species are often difficult to identify. Pathogenicity is governed by mobilisable plasmids. UK 2. 2.g. Ralstonia solanacearum is the sole pathogenic species. UK 3. Within the beta Proteobacteria. 3) plus fatty acid profiling are useful both in diagnosis and differentiation.2. W (2001) Laboratory guide for identification of plant pathogenic bacteria . 2. once almost all incorporated within Erwinia. Names of Plant Pathogenic Bacteria http://www. now has 8 genera proposed containing plant pathogenic species (Brenneria. 2. Xanthomonas now comprises some 20 species. biovar. Biovar and race determination are widely described (1. gyrase B gene. 2. et al. J. Pathovar determination does not always rely on prior determination of species and traditional methods for identification and diagnosis (1. The Enterobacteriaceae. Jones.B and Chun.A. 2. Bradbury. 2. Most produce fluorescent pigments.asp 23 . Erwinia. Traditional tests (1. Genus determination is largely based on 16SrDNA sequencing. 3) Diagnosis is often straightforward (2).isppweb.7 Gram Negative Bacteria.org/names_bacterial. tumefaciens. 3) are still very useful. Samsonia. within the gamma Proteobacteria There are 3 main groups. Gene sequencing best differentiates species. 3).F. Enterobacter. Lelliott. within the alpha Proteobacteria The main genus is Agrobacterium. D. The latter is the best but depends on gene sequencing. Genetic fingerprints such as repetitive sequence PCR perhaps best differentiate species and pathovars. Blackwell Scientific Publications.W. The species within each can be differentiated by traditional tests (1. (1986) Names of plant pathogenic bacteria. actually comprises many genomospecies at the 70% DNA homology level. Genus determination can be achieved by fatty acid analysis but species determination is best achieved by gene sequencing e. 2. which is not well studied. Pantoea. References 1.6 Gram Negative Bacteria. USA 4. J. N. syringae. radiobacter and A.. 3) Within the species. 3). many with pathovars. the main genera are Acidovorax. and Stead. Six species are recognised but it is known that biovar 1 corresponding to A. 3.boonham@csl. reporting back all hits.nlm.gov/gquery/gquery. York. The data is then shared among all these systems. number of databases that you can search against. taxonomy etc. In Europe you submit to EMBL.ncbi.3. the most useful methods are searching for individual sequences of interest i. papers. Primer design and Bioinformatics Neil Boonham Central Science Laboratory. The databases can be searched in a number of ways. using a keyword. This can be accessed in a number of ways. i. searching a database for sequences similar to a query sequence.fcgi) searches all possible databases. and in the US to the NCBI. 24 . UK n.1.nih. followed by a range of similarity based searches.1ENTREZ search ENTREZ search is a keyword search that allows you to search a range of databases including the nucleotide databases. where the information can be either retrieved by cut and pasting or saving to files in a range of formats.uk 3.e. a search for Pytophthora ramorum: Enter keyword here Papers Sequences Hyperlinks lead you through into the sequence accessions. a simple ENTREZ search (http://www.e. YO41 1LZ.gov. There are a large. in Japan to the DNA databank of Japan.1 Database searching The National Centres maintain a single database called the non-redundant database. A submission to any of the centres results in the permeation of the data into all the databases. Sand Hutton. and ever growing. For example. the Genbank file (the sequence accession) and FASTA format (for further analysis software).2 Retrieving sequence information Information can be removed individually following the hyperlinks.) as follows. or in groups by (a) highlighting the individual items. (a) Highlight accessions (b) Select format (c) Select output 25 . text etc.g. FASTA etc. For sequence information the most useful formats are the summary.) followed by (c) the form in which you want the information (e.g. Genbank. then (b) selecting the format (e. save to file.3. and BLAST 2 sequences. and other genomes. use the default setting to begin with for most searches (ignore the options section). microbial. thus negative results (no significant matches may be difficult to interpret). Remember the results only reflect what is present on the database. and tentative human consensus sequences.nlm.gov/BLAST) for comparing gene and protein sequences against others in public databases now comes in several types including PSI-BLAST.3. The simplest and most useful is a BLASTn or nucleotide-nucleotide BLAST. PHI-BLAST.3 BLAST searching The Basic Local Alignment Search Tool (BLAST) (http://www. Specialized BLASTs are also available for human. This returns information from the database about a query sequence. as well as for vector contamination. (a) Paste sequence here (b) Click on BLAST 26 . essentially allowing you to identify ‘unknown’ sequences or confirm the identity of a known sequence.nih.ncbi. immunoglobulins. Paste in your query sequence into the link. malaria. 3. The results will be displayed graphically (colour coding score values). followed by a list of highest scoring matches and finally alignments of the Highest Scoring Sequence Pairs (HSP’s).3.1 Interpreting the results After submitting a BLAST query a link will take you to the results by pressing the Format button for your request (again at this stage ignore the view options below). Sequence with highest score value Score value Probability Alignment for the highest scoring pair (HSP) 27 . html). In contrast a result of 0. The score is the basis of the BLAST search. A BLAST search is looking for regions of sequence that align together with a probability that is greater than random chance thus giving a high score value these are called High-scoring Segment Pairs (HSP).3. For each region of alignment all the scores are added up.1 means that there is a 1 in 10 chance of a random sequence of the same length generating this score value thus it is probably not significant. and can be found at (http://www. However it is important to understand the basics.3. When you get a BLAST result you also get a probability value. If there is no match the score is negative. This value is the probability that the HSP occurs by random. For example if you take a completely random sequence the same length as your query sequence there is a chance that you could get exactly the same sequence by random. This probability is based on the length of the query sequence and the total length of the database.2 Interpreting the score values Complete explanation of the interpretation of the score values is beyond the scope of this protocol booklet.nih.gov/BLAST/tutorial/Altschul-1. If you are comparing a short sequence it is more likely that a random sequence could give you the same result. 28 . A probability of 0 means that there is essential no chance your match was random. In an alignment of a pair of sequences each nucleotide is given a score depending on whether it matches or not.nlm. This is not very likely. A probability of 10-50 (reported as 1 E -50) means that there is 1 in 1050 chance a random sequence of the same length would generate this score value.ncbi. If you are comparing a sequence to a large database of sequences there is more chance that you have a random sequence in there that matches your query sequence than if you are comparing your sequence to a small database. and so most hits returned with a score of 10-50 are probably real. though the score can never go below zero. and if there is a match the score is positive. outputted to text and pasted into the alignment tool. since it allows you to identify regions of sequence that have conservation (perhaps between members of the same species) and divergence (perhaps between members of closely related species).ac.1 Results The results of an alignment are shown below and allow the identification of conserved and divergent sequence regions. Export to text a list of sequences in FASTA format and paste here Press run 3.4 Making a multiple sequence alignment Multiple sequence alignments allow you to identify nucleotides with identity to other sequences. It is also the first part of the process for generating phylogenies for deriving evolutionary relationships. In both cases the input format is a list of sequences in FASTA format that can be collected following an ENTREZ keyword search. It is also a useful tool to use prior to performing diagnostic PCR primers. 29 .uk/clustalw/).3.4. The most commonly used algorithm is CLUSTAL and can be performed at several web sites (e.ebi. http://www.g. accept the default settings to begin with. RTF format that can be viewed in Word. In this version an .embnet.html.org/software/BOX_form. 30 .ch.ALN format file is produced by CLUSTAL W that is inputted (by cut and pasting) to give an output file in an .Divergent nucleotide positions The alignments can be highlighted using a program such as BOXSHADE on http://www. 31 . when it comes to either exploiting sequence differences for discrimination or coping with sequence differences for universal amplification. Although there are no universal rules for primer design and many people use different approaches several generalities can be borne in mind. where sequence differences should be placed in the 5´ end where they will have little effect whilst the 3´ end should be sited in the most conserved region of sequence. Clearly all of these factors are linked together. targeting the correct region of sequence. For diagnostic work an amplicon (PCR product) size of between 200-500bp in length gives a balance of good amplification efficiency (smaller products amplify more efficiently) whilst being relatively easy to size following agarose gel electrophoreses.3. With this in mind the primer can be conveniently split into 3 regions. PCR primers are usually 18-25nt in length with a GC content of between 40-60 % and a melting temperature (Tm) range of between 55-65ºC.e. whilst differences in the 5´ will have little effect on discrimination of closely related sequences. From a diagnostic point of view specificity is generally the most important aspect of primer design. The converse is true for universal detection. 5´ 3´ Least Important < Less Important < Most Important For discrimination the sequence differences should be in the 3´ end of the primer. i. thus selecting the required Tm in primer design software should account for the other parameters.5 Primer design theory When a diagnostic sequence has been found (often called a molecular marker) primers can be designed to it using a primer design tool. the most useful are as follows.mit. It is a good idea to check the specificity of the primers and the amplicon by BLAST searching with the sequences. All primer sets must be tested experimentally for specificity and sensitivity.edu/cgi-bin/primer3/primer3_www. This package has a large number of preset defaults and options to customise the design to meet your requirements.cgi). 32 . both free and commercial.6 Primer design practice A large amount of software is available for primer design. for example (http://frodo. Following PCR it is necessary to sequence the amplified products of the correct size. small is best for diagnostic use Default parameters for primers – these are ideal for most situations The program outputs an optimal (most closely fits the program parameters) primer pair followed by some further possible designs.3. Paste sequence here Indicate the position of the 3′ end of the primer Product size. and confirm the identity by BLAST searching.wi. for general-purpose use many of the free packages are effective. gov. Manipulation of the magnetic beads in kits such as the ChargeSwitch gDNA Plant kit can be performed manually using devices such as the BioNobile PickPen.tomlinson@csl. PickPen 8-M (BioNobile). ChargeSwitch Technology for DNA extraction. Isolation of bacterial DNA Jenny Tomlinson Central Science Laboratory. York. or can be automated. right.4. Many of these use magnetic silica beads. the ChargeSwitch gDNA Plant kit (Invitrogen) uses magnetic beads coated with a material with pH-dependent charge (Figure 1). 33 . washing the particles to remove contaminants.uk 4. UK j.1 DNA isolation using ChargeSwitch Technology (CST) Several DNA extraction kits are available that work on the principle of binding DNA to magnetic particles. Charge Switch DNA extraction method Lyse sample with SDS + Low pH ChargeSwitch magnetic beads have positive charge at low pH: nucleic acid binds High pH Charge is neutralised >pH 8. Figure 2.5: DNA is instantly released Bind DNA to CST beads pH <6 Wash beads + bound DNA pH = 7 Elute DNA from beads pH = 8. KingFisher mL (Thermo) for automated handling. for example using the KingFisher mL (Thermo) (Figure 2). In contrast. Devices for manipulation of magnetic beads for DNA extraction: left. and finally eluting the DNA from the particles. YO41 1LZ. Sand Hutton.5 Figure 1. which bind DNA in the presence of chaotropic salts. and run programme gDNA (approximately 10 minutes). Add 900µl Lysis Buffer to 100µl sample. 34 . 9.irritant Detergent: Triton X-100 – irritant. plus appropriate plastic ware (strips and combs) Microcentrifuge Pipettes (200µl and 1000µl) Sterile filter pipette tips Gloves Microfuge tubes Ice Method 1. 5. 2. Chill the Precipitation Buffer on ice. harmful if swallowed You will also need: KingFisher mL. For each sample set up a KingFisher mL strip containing: Well 2: 1ml Wash Buffer Well 3: 1ml Wash Buffer Well 4: 1ml Wash Buffer Well 5: 200µl Elution Buffer 8. 6. Insert new tip combs on the Kingfisher mL.ChargeSwitch / KingFisher DNA extraction protocol Reagents required: ChargeSwitch gDNA Plant kit (Invitrogen. then add 40µl CST Beads and 100µl 10% Detergent and mix gently by pipetting. Transfer 1ml of clarified sample to a fresh microfuge tube. Transfer the sample and beads to Well 1 of the KingFisher mL strip. 3. Transfer the eluted DNA to a clean labelled microfuge tube. 7. Add 100µl SDS. 4. 10. harmful if swallowed Precipitation Buffer: contains potassium acetate and potassium chloride . cat # CS18000) Safety Information: Please take care when using the following reagents: Lysis Buffer: contains Urea – irritant SDS: sodium dodecyl sulfate – irritant. Resuspend the CST Beads thoroughly before use. then incubate at room temperature for 5 minutes. Store the DNA extracts at -20°C. Add 400µl cold Precipitation Buffer and vortex to mix. Centrifuge at approx 12 000 x g for 5 minutes. YO41
[email protected] Conventional PCR PCR utilises short DNA sequences (primers) to amplify a specific target DNA sequence. Since the extension products are also complementary to and capable of binding primers. UK r.e. Sand Hutton. 35 . In conventional PCR these copies are observed by running the reaction mix through an agarose gel.5. Each probe. Real-time analysis also facilitates quantification of the amount of sample DNA present in the reaction by ascertaining when (i. during which PCR cycle) fluorescence in a given reaction tube exceeds that of a threshold (Threshold Cycle (CT)). designed to hybridise specifically to the target PCR product (between the two primers). Ethidium bromide stained gel (under UV illumination) showing PCR products of 224 bp.gov. 1. effectively doubling the amount of the target DNA sequence. Fig. 5. separating the dyes. Comparison between reaction tubes and / or known standards can quantify the amount of DNA template present in a given tube. Lower CT values indicate higher amounts of target DNA (Fig 3).2 Real time PCR Real-time (or TaqMan) PCR exploits the 5′ nuclease activity of Taq DNA polymerase in conjunction with fluorogenic DNA probes. York. is labelled with a fluorescent reporter dye and a quencher dye. proceeds across the target sequence. Repeated cycles result in an exponential increase in target DNA to a point where there are enough copies of the sequence for it to be visualised. During PCR amplification the probe is digested by Taq DNA polymerase. and resulting in an increase in reporter fluorescence (Fig 2). by Taq DNA polymerase. PCR and real-time PCR protocols Richard Thwaites Central Science Laboratory. Ethidium bromide chelates to DNA and fluoresces under UV illumination (Fig. The gel is then stained with ethidium bromide. Each primer hybridises to opposite strands of the target sequence and are orientated so that DNA synthesis. 1). the cycle can be repeated after a denaturation step.uk 5. Repeated PCR cycles result in exponential amplification of the PCR product and corresponding increase in measurable fluorescence intensity (usually measured by a laser present within the real-time PCR machine. are produced from positive reactions. other real-time PCR chemistries exist. Fig. Real-time PCR (TaqMan) chemistry Ralstonia solanacearum : Enrichment Taqman PCR for 96. 72. though a postPCR melt-curve analysis is required to ensure that the target PCR sequence has been amplified. and therefore fluorescence. Typical amplification plot showing increase in fluorescence from one target DNA molecule at different concentrations in the initial samples 36 . As the PCR progresses increased amounts of DNA. 3. This can be analysed in the same way as fluorescence emitted during TaqMAn real-time PCRs. 2. These include the use of DNA chelating dyes such as SYBRGreen which binds non-specifically to DNA and emits fluorescence under excitation by a laser. Fluorescence absorbed Primer TAQ Q R Probe Primer R TAQ Q Probe Cleavage Polymerisation and 5’ Nuclease activity Fig. 48.Although TaqMan chemistry has been described here. 24 and 0 hours growth in SMSB for 106 cells per mL of potato extract. This approach to genomic fingerprinting is referred to as rep-PCR and offers a highly sensitive level of analysis.5. especially Gram-negative bacteria. Nucleotide sequence determination of these repetitive regions has enabled the design of PCR primers specific to each region. 37 . enterobacterial repetitive intergenic consensus (ERIC) sequences and the BOX element. PCR with these primers thus give rise to amplification products that reflect each number and distribution of repetitive sequences. These are referred to as repetitive extragenic palindromic (REP) sequences. suitable for species and in some cases pathovar / biovar identification. Three DNA families have been recognised that are unrelated at the DNA level.3 rep-PCR Characterisation of bacterial genomes has led to the recognition of repeated DNA sequences that are conserved within diverse bacteria. 4 Protocols 5. PCR reaction mix Reagent Sterile UPW 10x PCR buffer MgCl2 (25 mM)) d-nTP mix (2.CCG GTG GGC TTG GCG CCG .63 U 25.0 0. corylina and fragariae.CCG GAA ACC GGC AAG AAG GCA .GCG TGC CGC AGC CGC . Further optimisation of this programme may be required for use with other real-time PCR systems. 38 .3' Probes Xaf pep-P 5’ . Note: This programme has been optimised for use with the ABI 7700 and 7900 sequence detector TaqMan systems and the Cepheid Smartcycler system. 2007.125 4.75 0.875 2.3' The real-time PCR with Xaf primers and probes detects strains of Xanthomonas arboricola pathovars pruni.75 0.5 0.) Primers and Probes Primers Xaf pep-F Xaf pep-R 5' .5 3.5 mM 200 µM 300 nM 300 nM 100 nM 0.3' 5' .5 2.4.5.1 Real-time PCR protocols for Xanthomonas arboricola (Weller et al.5 mM) Forward Primer (10 pmol µl-1) Reverse Primer (10 pmol µl-1) Probe (5 pmol µl-1) Taq polymerase (5U/µl) Sample volume Total volume : Quantity per reaction (µl) 10.0 PCR Reaction conditions Run the following programme: 1 cycle of: (i) 10 minutes at 95 °C (denaturation o f template DNA) 40 cycles of: (ii) 15 seconds at 95 °C (denaturati on of template DNA) 60 seconds at 60 °C (annealing of primers) (annealing time can be reduced to 30 seconds if using the Smartcycler system).0 Final concentration 1x 3. 7 mM MgCl2) 1.75 µl 3.5 µl 6.5% agarose gel (20 x 24 cm) in 1 x TAE buffer. 39 .0 U 25.0 µl REP-PCR reaction conditions 95OC - 2 min 94OC 92 OC 40OC 65OC - 3 sec ) 30 sec ) 60 sec ) 8 min ) 65OC 4OC - 8 min indefinitely x 35 cycles Electrophoresis conditions It is very important to standardise electrophoresis conditions as much as possible in order to minimise variation in band patterns between gels.one at each end and one in the middle. Variation between gels will affect the quality of the analysis performed post-electrophoresis.4 µl 1.2-2 µl of 6 x loading buffer (LB) and add the final mix into the wells on the gel. Therefore care must be taken to follow the same protocol for each gel / run performed.75 µl 0. On a strip of parafilm add 6-10 µl amplified DNA mixed with 1.0 µl Total volume : Final concentration 1x (6.5.4.2 µl 2.7 µl 1. Leave three of the wells for the molecular weight marker . Run the gel at a constant voltage of 105V (approx 55 mA) per gel.25mM 4 µg 10% 3 µM 3 µM 2.95 µl 2.25 µl 0. Set the powerpack to run for 5-6 hours at room temp (or 70V/~23 mA for 16-18 hrs at 4 ºC). Prepare a 1.2 rep-PCR for genomic fingerprinting Schaad (2001) Primers REP1R-1 REP2-1 5’ III ICG ICG ICA TCI GGC 3’ 5’ ICG ICT TAT CIG GCC TAC 3’ PCR reaction mix Reagent Sterile UPW 10x PCR buffer MgCl2 (25mM) d-nTP mix (25mM each) BSA (20 mg per ml) DMSO Primer REP1R-1 (20µM) Primer REP2-1 (20µM) Taq polymerase (5U/µl) Sample volume Quantity per reaction 2.5 µl 3. Use a 20 or 30 tooth comb (1 mm). 0 µl Total volume: Final concentration 1x (6.75 µ 0.2-2 µl of 6 x loading buffer (LB) and add the final mix into the wells on the gel.2 µl 2.7 mM MgCl2) 1. Therefore care must be taken to follow the same protocol for each gel / run performed.5 µl 3.0 U 25. 600ug/1000ml) CARE! ETHIDIUM BROMIDE IS MUTAGENIC .wear latex disposable gloves. Prepare a 1.7 µl 1. After electrophoresis. Variation between gels will affect the quality of the analysis-performed post-electrophoresis.0 µl ERIC-PCR reaction conditions 95OC - 94OC O 92 C 50OC 65OC 65OC 4OC - 2 min 3 sec ) 30 sec ) 60 sec ) 8 min ) x 35 cycles 8 min indefinitely Electrophoresis conditions It is very important to standardise electrophoresis conditions as much as possible in order to minimise variation in band patterns between gels.3 ERIC-PCR for genomic fingerprinting Schaad (2001) Primers ERIC1R ERIC2 5’ ATG TAA GCT CCT GGG GAT TCA C 3’ 5’ AAG TAA GTG ACT GGG GTG AGC G 3’ PCR reaction mix Reagent Sterile UPW 10x PCR buffer MgCl2 (25mM) d-nTP mix (25mM each) BSA (20 mg per ml) DMSO Primer ERIC1R (20µM) Primer ERIC2 Taq polymerase (5U/µl) Sample volume Quantity per reaction 2. Leave three 40 .5 µl 6.25 µl 0.25mM 4 µg 10% 3 µM 2. Use a 20 or 30 tooth comb (1 mm). 5.75 µl 3. On a strip of parafilm add 6-10 µl amplified DNA mixed with 1.4. switch off the power pack and remove the gel from the tank. Stain in ethidium bromide (final conc.4 µl 1.95 µl 2.5% agarose gel (20 x 24 cm) in 1 x TAE buffer. Stain for 30 mins and destain in water. 7 µl 1.0 U 25. Stain for 30 mins and destain in water. switch off the power pack and remove the gel from the tank.25 µl 0. 5.2 µl 2.of the wells for the molecular weight marker . 600ug/1000ml) CARE! ETHIDIUM BROMIDE IS MUTAGENIC . After electrophoresis.75 µl 0.7 µl 2.5 µl 6.7 mM MgCl2) 1.4.4 µl 1.5 µl 3. Run the gel at a constant voltage of 105V (approx 55 mA) per gel.4 BOX-PCR for genomic fingerprinting Schaad (2001) Primers BOXAIR 5’ CTA CGG CAA GGC GAC GCT GAC G 3’ PCR reaction mix Reagent Sterile UPW 10x PCR buffer MgCl2 (25mM) d-nTP mix (25mM each) BSA (20 mg per ml) DMSO Primer BOXAIR (20µM) Taq polymerase (5U/µl) Sample volume Quantity per reaction 6. Set the powerpack to run for 5-6 hours at room temp (or 70V/~23 mA for 16-18 hrs at 4 ºC).wear latex disposable gloves.25mM 4 µg 10% 3 µM 2.0 µl BOX-PCR reaction conditions 95OC - 2 min 94OC 92 OC 50OC 65OC - 3 sec ) 30 sec ) 60 sec ) x 35 cycles 8 min ) 65OC 4OC - 8 min indefinitely 41 .0 µl Total volume : Final concentration 1x (6. Stain in ethidium bromide (final conc.one at each end and one in the middle. Leave three of the wells for the molecular weight marker . Stain for 30 mins and destain in water. After electrophoresis.2-2 µl of 6 x loading buffer (LB) and add the final mix into the wells on the gel. On a strip of parafilm add 6-10 µl amplified DNA mixed with 1. 42 . 600ug/1000ml) CARE! ETHIDIUM BROMIDE IS MUTAGENIC . Run the gel at a constant voltage of 105V (approx 55 mA) per gel. Therefore care must be taken to follow the same protocol for each gel / run performed. Stain in ethidium bromide (final conc.5% agarose gel (20 x 24 cm) in 1 x TAE buffer. Set the powerpack to run for 5-6 hours at room temp (or 70V/~23 mA for 16-18 hrs at 4 ºC). Variation between gels will affect the quality of the analysis-performed post-electrophoresis.one at each end and one in the middle. Prepare a 1.wear latex disposable gloves. Use a 20 or 30 tooth comb (1 mm).Electrophoresis conditions It is very important to standardise electrophoresis conditions as much as possible in order to minimise variation in band patterns between gels. switch off the power pack and remove the gel from the tank. by realtime PCR. 1994. D. J.References Louws FJ. Plant Disease 83. 1991.E. Elphinstone. 43 . Schaad.. K-H. European Journal of Plant Pathology 106. Sechler. 2853-2858.. R. Detection of Ralstonia solanacearum strains using an automated and quantitative flourogenic 5’ nuclease TaqMan assay. European Journal of Plant Pathology 108.G.A.. and Boonham. Versolavic J. Fulbright DW. 2002.. Y. Stephens CT. Nucleic Acids Research 24: 6823-6831. Berthier-Schaad. 2007. R.J. Koeuth T.. S. A. N. Detection of Clavibacter michiganensis subsp.. H. 2000. sepedonicus in potato tubers by BIO-PCR and an automated real-time fluorescence detection system. Applied and Environmental Microbiology 66. Journal of Microbiological Methods 70. Elphinstone. J. D.. N.. Detection of Xanthomonas fragariae and presumptive detection of Xanthomonas arboricola pv. 2000. 1999. fragariae. Hall. Lupski JR. Elphinstone. Distribution of repetitive DNA sequences in eubacteria and application to fingerprinting of bacterial genomes. Weller. J. 379–383. Specific genomic fingerprints of phytopathogenic Xanthomonas and Pseudomonas pathovars and strains generated with repetitive sequences and PCR. Weller. 155–165. Smith. N. N.. J.A. Detection of Clavibacter michiganensis subsp.G. and Pukall. sepedonicus in potato tubers by multiplex PCR with coamplification of host DNA. DeBruijn FJ. Thwaites.. Pastrik. 1095-1100. Sequence analysis and detection of Ralstonia solanacearum by multiplex PCR amplification of 16S-23S ribosomal intergenic spacer region with internal positive control. K. Beresford-Jones. and Knorr. W. Applied and Environmental Microbiology 60: 2286-2295..G. S. 831-842 Pastrik. Stead. Parkinson. from strawberry leaves. 0 2) 5 x loading buffer 5 to 8 mg bromophenol blue in 10 mls 50% glycerol.APPENDIX 1) 50 x TAE buffer 242 g Trizma base (Sigma T-840) 18. BECAUSE GLYCEROL IS VERY VISCOUS.1 ml glacial acetic acid Make up to 1 litre with distilled water.6 g diSodium EDTA 57. Check pH is 8. CUT THE TIP OF A 5 ML PIPETTE TIP BEFORE WITHDRAWING 5 MLS GLYCEROL FROM THE BOTTLE AND ALLOW TIME FOR IT TO FILL UP AND TO DRAIN INTO A UNIVERSAL BOTTLE. 44 . This is a book primarily about identification. much time and effort can be saved in determining the cause of a plant disease. (2001). YO41 1LZ. • Diagnosis is the process of determining the causal agent of a disease. The most important pieces of diagnostic information are the symptoms themselves and with an experienced eye. York. which will almost certainly take more time than is necessary. the more cost effective the service becomes.e. A confirmatory diagnosis requires a host test (to complete Koch’s postulates) although.uk 6. The diagnostic requirements or level of diagnosis needed will depend on the value of the diseased crop or consignment. The starting point is having a pure culture. As a visual infection develops. There is a common tendency to isolate the bacterium and then start the process of identification.e. the symptomatic tissue may contain the pathogen but often in a decline 45 . molecular methods are getting close to replacing the need for a host test in some situations. another process open to unnecessary complication and creation of more work than is necessary. Rapid diagnosis is needed to allow appropriate control.g. It is highly cost effective to know what bacteria/other pathogens can cause a particular type of symptom in a host. Stead Central Science Laboratory. 100% chance of being correct. increasingly. The diagnosis may be presumptive i. • Identification is the process of allocating a name to an unknown bacterium. imported seed potatoes with high disease incidence. The more diagnoses that can be completed by a diagnostician in a day. UK d. a presumptive diagnosis may be made at this point. A Presumptive diagnosis usually comprises isolation plus preliminary identification of some sort. This is a book primarily about diagnosing the diseases.) by Schaad et al. or confirmatory i.6. Methods for the diagnosis of bacterial diseases of plants by Lelliott &Stead 1987. These include: • • Laboratory guide for identification of plant pathogenic bacteria (3rd ed. Detection can give a presumptive diagnosis The approach to these issues can be seen in the various books written on diagnosis and identification. There may be less value in confirming the cause of a single leaf spot in a field than a consignment of high value. Detection is often based on a single test/method e. • Detection is the process of demonstrating the presence of a specific bacterium in a sample. with around 90% chance of being correct. IF or PCR. There are usually not many that can. The time taken to achieve a diagnosis is often critical.1 Diagnosis or Identification: Detection or Diagnosis These terms are often used wrongly! If they are fully understood and used
[email protected]. Minimum requirements for diagnosis David E. Your diagnostic strategy is now simply to eliminate those which are not involved based on the minimum amount of information The next stage is to isolate the bacterium. Sand Hutton. and ability to grow at selected temperatures. The LOPAT class gives perhaps as much diagnostic information as any modern method. are rapid. this will depend on your experience with the disease and the likely pathogens. syringae. it may be better to discard it and start again. Even if it has. However it is important to understand the significance of the tests applied. pH or salt concentrations. than to attempt to identify all colony types in the hope that you may have the pathogen somewhere on the plate. Your lab may have decided to introduce a single standardised identification system such as fatty acid analysis or gene sequence based identification. that zone that is either side or usually just in front of the actual edge is where you are most likely to find viable pathogen and usually in the absence of competitors. Of the major diseases covered in this COST action. elevation. there is benefit in selecting key simple tests that help eliminate those possible pathogens you might expect to cause the symptoms seen on the host. The leading edge of the symptomatic tissue. a pure culture with a typical morphology and perhaps one or two other key tests. Until recently there was no cost effective and relatively simple way of allocating xanthomonads to species. pectolytic ability. yyyyy belonged to X. then the process of further characterisation or identification starts. In these days of exciting molecular methods we often ignore such valuable diagnostic information as pigmentation (soluble or insoluble). In this case. For example. colony size. arginine dihydrolase activity. Whereas all strains of a species are likely to be either oxidase positive or negative. you may also decide to make a presumptive diagnosis. edge. Species identification was based on the inference that pv xxxx belonged to X. then a negative oxidase test could clinch it or at most a LOPAT group 1. savastanoi pv. Your skill at selecting the correct leading edge is defined usually by the purity of the isolation you make. most can be effectively diagnosed presumptively based on symptoms. Only if this were the first case of halo blight in your country or the consignment was worth a huge amount of money and was destined for destruction. and you go further and get a pure culture of a blue fluorescent bacterium on King’s B medium. These include oxidase test. If you are still not convinced. Successful pathogen isolation is often indicated by a pure culture of a bacterium with high colony numbers on a streak plate. would a confirmatory host test likely to be required. Many of the traditional tests. A simple Gram stain will usually differentiate Gram positive and negative bacteria. it matters not whether the bacterium belongs to the species P. perhaps only 80% of strain of a given species might be able to utilise a particular sugar For fluorescent pseudomonads the LOPAT tests are very good examples of the use of simple tests. If your plate is very mixed. but this is not always the case. The presence of cytochrome c oxidase as in the oxidase test has greater evolutionary significance than the abilty to utilise mannose rather than galactose.phase of growth or in competition with secondary invaders. which additionally indicates a hypersensitive response in tobacco and thus a pathogen. shape. Assuming you have not been able to make a presumptive diagnosis at this stage. cheap and simple and often allow cost effective elimination of the contenders. This too is a very important step in the diagnostic decision process and if your isolate is pure and typical of one of the likely causal agents. phaseolicola can cause those symptoms and make a presumptive diagnosis. surface texture. arboricola whereas pv. savastanoi or to P. If you are not entirely happy with this. Again. The main problems may arise 46 . that rarely vary within strains of a given species. so rarely performed these days. if you are presented with a bean leaf showing typical yellow haloes you may decide that only P. Thankfully it seems that gene sequencing can now give classifications that agree with those based on complex DNA homology. your presumptive diagnosis may be made here. hortorum. 47 . It is then that the molecular methods such as gene sequencing and repetitive sequence PCR come into their own.with the complexity of fluorescent pseudomonads for example on Corylus avellanae and Prunus spp and possibly confusion of xanthomonads with similarly pigmented members of the Pantoea agglomerans (Erwinia herbicola) complex. This method is widely regarded as one of the best methods for rapid. Bacteria alter their lipid fatty acid composition to maintain membrane fluidity under varying environmental conditions.stead@csl. Automated identification is facilitated by comparison with libraries (databases) of profiles produced under standard conditions. Pathovars in some species. are often not well differentiated. family and genus. It is essential therefore to maintain strictly controlled growth conditions to ensure consistent and reliable results.500 aerobic and anaerobic bacterial species. which has proven to be an important and cost effective tool.1 Summary Fatty acid profiles are well established for classification and identification of plantassociated bacteria. Acidovorax. Profiles are based on standardised culture. but not all bacteria. Fatty acid profiling David E. Many Gram-negative genera have unique series of hydroxy acids. Ralstonia. Most bacteria have between 5 and 30 of these acids.50% (usually 5 . To meet this requirement we culture the cells under strict conditions and then harvest them for analysis purposes.7.2 Introduction One method of identifying bacteria.g. Accuracy of identification can be excellent 7.10% dry weight) which can be extracted. we need to obtain sufficient numbers of cells (approximately 40 mg). occur in bacteria. Sand Hutton. is fatty acid profiling. Burkholderia.uk and j. Fatty acid profiles do not tend to support genomospecies within Pseudomonas syringae but for most other species correspond well with DNA and rRNA homology. Bacterial membranes contain lipids in concentrations of 0.gov. Pseudomonas. a library generated in house by the National Collection of Plant Pathogenic Bacteria (NCPPB) in which almost all known plant pathogenic bacteria and closely related bacteria are housed. YO41 1LZ. e. belonging to several classes. Pseudomonas syringae.uk 7. and analysed by gas chromatography quantitatively and qualitatively. These can be selfgenerated or purchased.2 . Rhodococcus. subspecies and sometimes biovar and pathovar level. In order to analyse and identify an isolate of a bacterium. Xanthomonas and Xylophilus all have characteristic profiles. This system allows for identification of many. It also uses the NCPPB3 library.gov. accurate and inexpensive identification of bacteria. Over 100 fatty acids of between 8 and 20 carbon atoms length.heeney@csl. and species to species. Once the 48 . simple chemical extraction and gas chromatographic separation of fatty acid methyl esters (FAMEs). Gram-positive genera usually lack hydroxy acids but are rich in branched acids. Stead and John Heeney Central Science Laboratory. converted to fatty acid methyl esters (FAMEs). UK d. The types occurring are usually indicative of the class. FAMEs are identified and quantified to give a profile for each strain. The relative amounts of individual fatty acids present often allow accurate differentiation at species. Agrobacterium. York. but in some cases it is also unique at biovar or pathovar level. The type and percentage of individual fatty acids present in bacteria not only varies from genus to genus. Clavibacter. It uses the Sherlock® Microbial Identification System which is used worldwide in clinical and environmental laboratories to identify over 1. anteisobranched (17:0 anteiso). This software enables comparison with a commercially available library (TSBA6) and an in-house generated library (NCPPB3). in turn. The TSBA6. the presence of 13: 0 iso 3OH is virtually exclusive to Xanthomonas. and histoplots showing the values of the sample compared to the nearest library entries. cyclopropanes (17:0 cycopropane). If calibration fails. This latter library is based on the cultures held in the National Collection of Plant Pathogenic Bacteria (NCPPB). The GC uses a commercially available calibration standard. and analysed.0 commercial library is based on cells cultured for 24 hours. straight chain mono-unsaturated (16:1 w7cis). The Hewlet Packard HP6890 series GC is controlled by HP Chemstation software. Once the sample has been analysed a profile is produced which identifies and quantifies each fatty acid as a percentage of the total peak area.isolate has been cultured and harvested. which is analysed twice prior to each set of runs and is also analysed once at set intervals during the run sequence.3 FAMEs FAMEs are fatty acid methyl esters of the fatty acids released from cellular lipids. converted to fatty acid methyl esters (FAMEs). the fatty acids can be extracted. iso-branched (15:0 iso). which are generally 820 carbon molecules in length. Fatty acid profiling relies on separating the fatty acids according to their size and conformation. primarily straight chain saturated (16:0). This. There are several major classes of fatty acids in bacteria. Samples are analysed. Most bacteria contain between 5 and 30 fatty acids. the run automatically stops. A comparison is made to the library entries and a profile is produced complete with a similarity index. 7. Reference can also be made to a commercially available library (TSBA6. Gram positives have branched acids and rarely have hydroxy acids. the resulting profiles are compared to the libraries and an analysis report is automatically generated. Table 1 shows the diagnostic patterns of hydroxy acids for various genera of Proteobacteria (Gram negatives) 49 . They are converted to FAMES to make them more volatile for gas chromatographic analysis. An experienced operator can prepare many samples in a day. cells are cultured for 24 hours or 48 hours depending on species and genus. hydroxy (12:0 3OH) and mixed (13:0 iso 3OH or 17:1 cyclopropane) Gram-negative bacteria invariably have hydroxy acids and rarely have branched acids. interfaces with Midi Sherlock software. For the NCPPB library. When used in conjunction with selected traditional and more recently developed diagnostic methods it can be a very informative diagnostic tool. leave the machine running overnight and obtain results the following day.g. although it should be noted that conditions for culturing are not exactly the same as those used for the NCPPB reference organisms. In contrast. Some genera have rare acids e. which represents nearly all known plant pathogenic taxa. The resulting fatty acid profiles can then be compared to a library of NCPPB reference organisms to help determine the identity of the bacterial sample under analysis. This procedure uses a Hewlet Packard HP6890 series Gas Chromatograph.0). For Proteobacteria (Gram negatives). accuracy of identification can only be as good as the quality and content of the library. Although the initial costs of equipment and software is high. We use 24 hour growth on Trypticase Soy Agar (TSA) at 28 ºC and harvest c. Similarity indices of less than 0. As well as the match.5 even though they may be first choice in the library are rarely correct identifications. It must be remembered that. The organic phase is washed with sodium hydroxide. removed and injected into the gas chromatograph for analysis 5. Cells are harvested and boiled in sodium hydroxide and methanol to saponify the lipids in the cells 2. Likewise the first choice even with a high similarity index may not be the true identity but it should be closely related to it. Similarity indices of 0. the presence/absence of hydroxy acids can be diagnostic at genus level.7 or above are generally accurate at species level. The fatty acid methyl esters (FAMEs) are then transferred from the aqueous phase to organic phase in a mixture of hexane and tert butyl ether 4. For fastidious pathogens incubation time can be extended but must be the same as used in the library development. running costs are low. The profile is printed off listing all the named peaks and their peak areas. Thus the method cannot replace the diagnostician but together with the other diagnostic information. The FAMES are then identified according to their equivalent chain length and quantified. If the species you have is not in the library it cannot be identified.7.4 Methods In order to obtain reproducible results. 40 mg growth from confluent growth avoiding the first (wash-out) and last (individual colonies) streaks. temperature and nutrients. though this is a generalisation (see Table 1). 50 . bacteria must be grown under uniform conditions of time. The resulting fatty acids are converted into methyl esters by heating in hydrochloric acid and methanol 3. Gram-negative and Gram-positive bacteria have different profile types with key types of fatty acid present or absent. fatty acid analysis can be a very cost effective and useful diagnostic method. if using a library. the chemotaxonomic information provided can be extremely useful. The extraction procedure has several steps: 1. This profile is then compared with the libraries and matches given as a similarity index. Differences between species within a genus or pathovars within a species tend to based on quantitative differences between the same FAMES rather than on the presence/absence of specific acids. Table 1. Characterisation of Proteobacteria genera by presence/absence of specific hydroxy acids FATTY ACID 10:0 12::0 12:0 14:0 16:0 16:0 16:1 18:1 11:0 13:0 3OH 2OH 3OH 3OH 2OH 3OH 2OH 2OH iso3OH iso3OH Alphaprot Agrobacterium + + (+) Betaprot Acidovorax + Burkholderia + + Ralstonia + + + + + + + Gammaprot ‘Erwinia’ Pseudomonas Xanthomonas + + + + + (+) + + . 51 . 1 Introduction Microbial identification based on partial gene sequence data is now a commonly used procedure. Sand
[email protected] Procedures 8. 3. a relatively greater or lesser difference between two species suggests a relatively earlier or later time in which they shared a common ancestor.gov. Use 2 µl of suspension as DNA template per PCR reaction. Store at –20 ºC until required.uk 8. The nucleotide base sequence of the gene which codes for 16S ribosomal RNA has become an important standard for the definition of many bacterial species. Suspend isolated bacterial colonies removed from pure cultures on agar plates in molecular grade water. 4.8. These methods offer increasingly higher levels of discrimination between closely related bacteria. UK n. York.2. 52 . The methods involve 3 stages: • PCR amplification of the target sequences • Purification and analysis of the amplified DNA • Sequencing of the amplified DNA 8. Heat 100 of suspension at 96 ºC for 4 min (Gram –ve) or 15 min (Gram +ve). Dilute in a further 900 µl of molecular grade water. 2. Identification of bacteria by partial gene sequencing Neil Parkinson Central Science Laboratory.1 Preparation of bacterial cultures for sequencing 1. For closely related organisms. Comparisons of the sequence between different species suggest the degree to which they are related to each other. YO41 1LZ. Rapid methods are described below for amplification of DNA from: • Partial 16S rRNA genes of most bacteria • 16-23S intergenic spacer regions of most bacteria • Gyrase B (gyrB) genes from Pseudomonas and Xanthomonas species. it may be necessary to analyse entire 16S gene sequences or to look at a number of different gene targets before sufficient variation in sequence is found to enable their discrimination. 5. Run PCR programme as follows: One cycle of : 95 ºC for 9 min Then 35 cycles of: 95 ºC for 1 min 55 ºC for 2 min 72 ºC for 1 min One cycle of: 72 ºC for 7 min Hold at: 4 ºC (Ensure that PCR machine is programmed for 50 µl reactions) 8.3 16-23S rRNA intergenic spacer region partial sequence (Pérez-Luz et al. 2004) 1.0 µl Primers: ufp1 AGT TTG ATC CTG GCT CAG (18 bp) urp1 GGT TAC CTT GTT ACG ACT T (19 bp) 2.. 4.PCR amplification of the target sequences Perform PCR reactions as required according to the following reaction conditions: 8.0 µl 19.5 min Then 35 cycles of: 94 ºC for 30 sec 60 ºC for 30 sec 72 ºC for 1 min One cycle of: 72 ºC for 5 min Hold at: 4 ºC (Ensure that PCR machine is programmed for 50 µl reactions) 53 .2 16S rRNA partial gene sequence (Weisburg et al. 4.0 µl 23sor 2. Add 2.grade H2O Fermentas 2X PCR Mastermix 25.0 µl urp1 2. 3. For each reaction prepare the following master mix: 16s14f 2.0 µl Fermentas 2X PCR Mastermix 25. 1991) 1. Add 2.2.0 µl Mol. For each reaction prepare the following master mix ufp1 2.0 µl Molecular grade H2O 19.0 µl DNA template into each tube.2.0 µl DNA template into each tube.0 µl Primers: 16s14f 23sor CTT GTA CAC ACC GCC CGT C (19 bp) TGC CAG GGC ATC CAC CGT G (19 bp) 2. Add 48 µl master mix per PCR tube. Run PCR programme as follows: One cycle of : 94 ºC for 2. 3.. Add 48 µl master mix per PCR tube. Run PCR programme as follows: One cycle of : 94 ºC for 2.5 Xanthomonas gyrB partial sequence (Parkinson et al.rsp1 CAA GGT GCT GAA GAT CTG GTC (21 bp) 2.75 µl 10x Long PCR buffer + Mg 2. 2000) 1. For each reaction prepare the following master mix: XgyrPCR2F 0.4 Pseudomonas gyrB partial sequence (Sarkar et al. 3. For each reaction prepare the following master mix: Forward primer 0.5 µl dNTP mix (2. Add 1.0 µl DNA template into each tube 4.0 µl Fermentas 2X PCR Mastermix 12.2. 4.875 µl Primers: XgyrPCR2F AAG CAG GGC AAG AGC GAG CTG TA (23 bp) Xgyr.gyr.75 µl Molecular grade H2O 10.2.125 µl Molecular grade H2O 17.75 µl X. Add 24 µl master mix per PCR tube. Add 24 µl master mix per PCR tube 3.. Run PCR programme as follows: One cycle of : 94 ºC for 5 min Then 30 cycles of: 94 ºC for 2 min 63 ºC for 1 min 72 ºC for 1 min One cycle of: 72 ºC for 7 min Hold at: 4 ºC (Ensure that PCR machine is programmed for 25 µl reactions) 8..5 µl Primers: Sarkar-GyrBf MGG CGG YAA GTT CGA TGA CAA YTC (24 bp) Sarkar-GyrBr TRA TBK CAG TCA RAC CTT CRC GSG C (25 bp) 2.0 µl Long PCR Enzyme Mix (Taq) 0. 2007) 1.75 µl Reverse primer 0.5mM each) 2.0 µl DNA template into each tube. Add 1.8.5 min Then 34 cycles of: 94 ºC for 30 sec 50 ºC for 45 sec 68 ºC for 1 min One cycle of: 68 ºC for 7 min Hold at: 15 ºC (Ensure that PCR machine is programmed for 25 µl reactions) 54 .rsp1 0. 13. Add 500 ml of Membrane Wash solution (mixed with ethanol according to the instructions with the Promega kit). www. 9. 1. Submit for sequence determination e.fsp. 2. Add 50 µl molecular grade water to the minicolumn and incubate at ambient temperature for at least 1 min. Centrifuge at maximum speed in a microcentrifuge for 5 min. Discard liquid in the collection tube. Discard liquid in the collection tube.g. 5.8. 5. 2. 4. Add an equal volume of Membrane Binding solution to the remaining PCR product (45 µl for 50 µl PCR or 20µl for 25µl PCR). Add 700 µl Membrane Wash solution (mixed with ethanol according to the instructions with the Promega kit). View bands under UV transillumination 8. Add 4 µl of 100 bp DNA ladder into the last well. Centrifuge at maximum speed in a microcentrifuge for 1 min.gyr. Transfer the minicolumn to a new 1.5 µl of purified DNA for a 50 µl PCR or 15µl for a 25 µl PCR to a new 1. to MWG in Germany (see web information at . 3. 12. 11.5 ml tube and spin with lids open in a speedvac vacuum centrifuge for about 1hr for 7.2. 6.7 Purification of PCR product If the PCR shows a single clear band the PCR product should be purified e.5 ml tube. 3. include 10 µl of the 17 bp internal forward primer X.short (GGC AAG AGC GAG CTG TA). 3.mwg-biotech. 10. Discard minicolumn and store purified DNA at –20 ºC until required.5µl.5 ml tube for sequence determination of each amplicon.g.6 Gel electrophoresis 1. 14. to allow evaporation of any residual ethanol. Stain the gel in ethidium bromide (600 µg per L) for 30 min and de-stain in water 6.8 Drying prior to sequencing 1. Include 10 µl of the forward primer in a separate labelled 1. 4. Centrifuge at maximum speed in a microcentrifuge for 1 min. using the Wizard SV gel and PCR clean-up system (Promega A9280) as follows: 1. 55 . 8.com). For Xanthomonas gyrB sequencing. Discard liquid in the collection tube.5h for 15µl. Centrifuge at maximum speed in a microcentrifuge for 1 min. Centrifuge at maximum speed in a microcentrifuge for 1 min. Mix 5 µl of each PCR product with 5 µl water and 2 µl 6X loading dye on parafilm and load into wells in the gel. 8.2.5 % agarose gel in 1X TBE buffer.2. 2. Place in an SV minicolumn in a collection tube and incubate for 1 min at ambient temperature. 7. Run for about 2h at 104 V. Prepare 1. Remove 7. J. Applied and Environmental Microbiology 70. D. and Catalan. V.. Heeney.S. D. J. and Stead.F. 2004.References Parkinson. Pelletier.. J.G. Identification of waterborne bacteria by the analysis of 16-23S rRNA intergenic spacer region. Barns. D. 1999-2012. abd Guttman. Yáňez. Evolution of the core genome of Pseudomonas syringae. D.A. 2007. Journal of Bacteriology 173. S. 191-204. M... S. S. 697-703. 16S ribosomal DNA amplification for phylogenetic study.M.. 1991. Bew. Phylogenetic analysis of Xanthomonas species by comparison of partial gyrase B gene sequences. Journal of Applied Microbiology 97. International Journal of Systematic and Evolutionary Microbiology 57.A. V. Aritua1. C. and Lane. a highly clonal endemic pathogen. N. 2881-2887.. W. Cowie. 56 . Pérez-Luz. Sarkar. Weisburg. 2004.. the conjugated antibodies appear as a fluorescent halo around the cells. It is recommended that the titre is determined for each new batch of antibodies.2) Disposable Eppendorph tubes Fibre free postlip paper Immersion oil (Sigma I-0890) Mounting medium (e. The titre is defined as the highest antibody dilution at which optimum reaction occurs when testing a suspension containing 105 to 106 cells per ml of the homologous strain of the target bacterium and using an appropriate conjugate according to the manufacturer’s recommendations. Direct IF uses the fluorescent compound conjugated directly to antibodies.A.) 9. Indirect IF firstly involves coating the target bacterium with the antibody. 57 .2 Material and equipment Polyclonal antisera produced in rabbit against target bacterium Anti-rabbit IgG FITC conjugate IF Buffer (PBS pH 7. YO41 1LZ. IF tests can be either direct or indirect. Zeiss Axioplan 2) Multispot microscope slides (e. When this conjugated antibody binds to the bacterium it’s presence can be visualised using a microscope with an ultraviolet (UV) light source and suitable filters.g. When bound to the target bacteria. (Essex) Ltd. During testing. Immunofluorescence cell staining for detection of bacterial pathogens Helena Stanford Central Science Laboratory. UK h.uk 9. the antibodies should be used at a working dilution(s) close to or at the titre. to bind to an antibody.1 Introduction Immunofluorecence (IF) is an accurate and useful serodiagnostic test which permits the target bacterium to be visualised under the microscope.3 Method Use validated antibodies.9. C. It is based on the ability of a fluorescent compound. York.g.gov. which specifically recognise the target bacterium. A second antibody is then used which is conjugated to the fluorescent compound and which recognises the first antibody. A simple example of an indirect IF protocol is given below: 9. which specifically recognises it. e. Vectorshield.g. The indirect IF approach usually results in a higher intensity of fluorescence around the target bacterial cells. Sand Hutton. Hendley. Vector laboratories) Humid chamber UV Microscope (
[email protected]. fluorescein isothiocyanate (FITC). 12H20 2.0g 58 . 3. Pipette 25 µl of the recommended dilution of an appropriate FITC conjugated (anti-IgG) antibody. Incubate as before for 30 minutes. Heat fix gently in a gas flame. Shake the droplets off the slide and rinse with PBS. 9. 4. Pipette 25 µl per window of antibody to the target bacterium (IgG). which are the size and shape of those on the positive control slide. Wash 3 times for 5 minutes in PBS and gently blot dry.2) Na2HPO4. Rinse and wash slides as before. 10. Prepare a separate slide with a known reference isolate of the target bacterium as a positive control (a suspension containing approximately 106 cfu per ml of the bacterium is usually used). 14. diluted in IF buffer to the pre-determined working dilution. 13. Place the slides on moist paper in a damp chamber for 30 minutes at ambient temperature. Scan around the periphery and transect the center both vertically and horizontally. whole cells. 12.0g Distilled H2O 1. Pipette approximately 10 µl of mounting buffer to each window and cover with a coverglass. 7.1. starting with the positive control slide. 11. Observe at high power under oil immersion (× 60 or × 100) starting with the FITC control. 8. 6. 5. Decimal dilution of sample Sample Duplicate of sample 1/1 1/10 1/100 1/1000 empty • • • • • • • • • • 1 2 3 4 5 6 7 8 9 10 2. Pipette 25 µl of IF buffer alone to the last two windows as an FITC conjugate negative control.7g NaH2PO4. IF Buffer (Phosphate buffer Saline pH 7.4g NaCl 8.2H2O 0. If no fluorescence is observed in the FITC control window. Prepare ten-fold dilutions of bacterial suspension or plant extract and pipette 25 µl of each dilution to duplicate microscope windows and allow to air dry. Look for brightly fluorescing. scan each test window for the presence of fluorescent cells with size and morphology typical of the target bacterium. Gently blot dry. EUPHRESCO – ERA-NET Leaflet 59 .10.