CSL COST873 TrainingManual

Short term Training Mission

Plant Bacteriology

MANUAL

3rd 7th March 2008

Central Science Laboratory

York, UK

European Science Foundation COST OfficeCOST OfficeCOST OfficeCOST Office

149 avenue Louise P.O. Box 12 1050 Brussels Belgium Tel: +32 (0)2 533 38 00 Fax: +32 (0)2 533 38 90 E-mail enquiries: [email protected] Website: http://cost.cordis.lu R.P.M.861.794.916 Tribunal de Commerce de Bruxelles

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This manual was compiled by:

John Elphinstone David Stead Neil Boonham Jenny Tomlinson Richard Thwaites Neil Parkinson Helena Stanford Elspeth Steel

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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

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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

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C D

E F

G

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Symptoms caused by Xanthomonas arboricola pv pruni: A) Yellowing and tip burning on peach leaves (Alan L. Jones, CABI); B) Spotting on peach fruit (Alan L. Jones, CABI); C) Cankers on peach twigs at bud break in spring (Alan L. Jones, CABI); D) Spotting on nectarine fruit (Alan L. Jones, CABI); E) Cankers and gumming on cherry tree (G M Balestra, University of Tuscia); F) Symptoms on Stanley plum fruit (Alan L. Jones, CABI); G) Pitting and gumming on nectarine fruit (Alan L. Jones, CABI); H) symptoms on cherry fruit (G. M. Balestra, University of Tuscia).

1.1. 2. Bacterial blight of walnut (Xanthomonas arboricola pv. juglandis)

Bacterial blight of walnut is a common, widespread and sometimes serious disease in most walnut producing areas. The disease is characterised by black lesions, angular to irregular on leaves, long and narrow on young stems and irregular often large on fruits.

A B

H

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Pictures L Gardan, INRA Symptoms of walnut blight: A) Canker on walnut twig; B) Brown necrosis on green twig; C) Spots on new fruit; D) Sunken spots on new fruit; E) Transverse section of fruit showing necrosis.

C

D

E

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Image from S Sule Leaf spot on walnut

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1.1.3. Bacterial blight of filbert and hazel (Xanthomonas arboricola pv. corylina).

One of the most characteristic symptoms is necrosis of the emerging buds in late spring. Infected leaves show small, angular to irregular, brown or black spots. Black spots and streaks may be found on young stems and cankers may also be found on twigs and branches. Young green nuts may also show small, black, necrotic spots.

A B

C D

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Images coutesy of L Gardan, INRA Symptoms of bacterial blight on hazel: A) Bud destruction and dieback; B) Yellowing on young leaves; C) Leaf spot symptoms on hazel leaf; D) Canker with cracks on bud; E) Close up of angular leaf spots; F) necrosis with halo on young hazel nut.

1.1. 4 Leaf spot, shot hole and bacterial canker of cherry and plum (Pseudomonas syringae pv. syringae and P. syringae pv. morsprunorum)

As well as causing cankers on the stems and branches of stone fruit trees (cherry, plum, peach almond, apricot and many other Prunus spp.) the bacteria also infect leaves, shoots and fruits. The disease is most common on cherry and plum in Europe. There are strains specific for each host. Cherry strains are cultivar specific and at least 2 races are recognized. 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.

The disease caused by the morsprunorum pathovar has a well defined seasonal cycle with a winter canker phase alternating with a summer leaf disease. Cankers, which are not perennial, are formed in the autumn or winter, but do not increase much in size until the following spring, when they enlarge rapidly, killing large areas of green bark. Once blossom fall occurs the progress of cankers is arrested and populations of bacteria within the cankers decline and usually die out. At the same time the leaf infection phase occurs. Spur leaves tend to be resistant as soon as

E

F

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they mature, but young leaves on extension shoots are infected. However, 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.

The disease caused by the syringae pathovar has a somewhat different epidemiology. Cankers can be perennial and the bacteria overwinter in them as small populations. Rapid multiplication occurs in the spring, ooze is often produced and the bacteria spread to leaves by rain splash. Unlike P. syringae pv. morsprunorum bacteria gain access to woody tissue only via wounds in leaf scars and bark, from which new cankers arise.

Symptoms vary between host species. In cherry, trees of all ages are susceptible and most cankers are found at sites of leaf scars on fruiting spurs. This usually results in die-back of the spur but may occasionally spread to form a canker in the parent branch. Cankers can also be located on the branches, especially on the crotch and angles between 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. When a canker girdles a branch dieback will occur. In plum, cankers occur most frequently on stems and trunks often leading to death of mature trees. Cankers may extend the length of the stem and often appear as dark-coloured, linear depressions in the bark. Once the stem is girdled, death ensues. Gumming is less common and not so obvious as on cherry.

Spots on leaves are caused by both pathovars and are usually reddish-dark brown, rounded or angular, often coalescing to form large irregular necrotic spots, which may drop out to give a shot hole effect.

A B

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Symptoms of Pseudomonas syringae on cherry and plum. A) leaf spot and shotholes on sour cherry (Alan L. Jones, CABI); B) Spur dieback and necrosis of mid-vein on Stanley plum (Alan L. Jones, CABI); C) Shothole symptoms on plum (AgrEvo, CABI); D) Symptoms on cherry fruit (Alan L. Jones, CABI); Canker and gummosis on sweet cherry branch (Alan L. Jones, CABI).

C

D E

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1.1.5 Bacterial decline and canker on nectarine and peach (Pseudomonas syringae pv. persicae).

This is a disease primarily of peach and nectarine but myrobalan and Japanese plum may also be affected.

In autumn and spring, small 2-5 mm round to elliptical lesions, olive to dark brown, occur on shoots at nodes and on internodes. The pathogen can usually be isolated. In spring, the pathogen can also be isolated from root tissues below cankers. Infected root tissue shows no sign of invasion.

In spring, investigation of dark discoloration of bark on previous year's growth reveals extensive brown and water-soaked lesions in the cambium. The pathogen may be isolated from these. On main leaders and trunks, extensive brown lesions are found with no discrete margin to healthy tissue. These are brown and dry. There is no visible sign of these lesions which are only detected when withering of leaves signals the girdling of the branch. Alternatively, deep incisions into wood of apparently healthy stems reveals discoloured wood - tracing down to larger branches reveals canker developments. It is difficult to isolate the pathogen from these lesions.

Initially small, olive, 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'. On fruits, small, olive, water-soaked lesions appear initially. These can be associated with the exudation of gum. This symptom is negligible in peach. In favourable conditions, especially in nectarine, these spots continue to expand during the spring and can cause severe distortion to developing fruit.

A B

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Images courtesy of Landcare Research New Zealand Symptoms of bacterial decline and canker on nectarine. A) symptoms on nectarine trunk; B) Necrotic bark and cortex of nectarine branch; C) Infection and distortion of nectarine fruit.

1.1.6 Crown gall of almond, apricot, cherry, hazel, peach, pecan, plum and walnut (Agrobacterium tumefaciens).

Crown gall is a common disease affecting a very wide range of host plants. On most hosts leaves are unaffected, although occasionally galls are formed on petioles. Stems have initially neat, rounded, smooth or fissured galls, usually just above soil level. On perennial woody hosts, galls become more woody and fissured with age, can be more than 10 cm across and can girdle the stem. Roots have rounded galls similar to those on stems but usually much smaller.

C

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Crown gall on walnut (Image: Larry, W. Moore, courtesy of CABI)

Crown gall on flowering cherry (Image: Larry, W. Moore, courtesy of CABI)

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1.2 Isolation and identification of bacterial pathogens of nuts and stone fruits

1.2.1 Isolation of Xanthomonas and Pseudomonas from cankers

1. Wash cankers in running water. 2. 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. Lift the epidermis with the pint of the scalpel and with blunt nosed forceps; tear it back to expose the leading edge of the lesion. Dissect out part of the edge with a flamed scalpel and tease it out in sterile water. Leave to stand for at least 30 minutes.

3. Examine microscopically for Gram negative, short, rod-shaped bacteria. The pathogen may only be present in small numbers or in reasonable numbers only in small pockets of the tissue of dormant cankers. If bacteria cannot be detected immediately, it is often necessary to examine a number of the lesions microscopically to find these pockets, and even then their numbers may be too low to detect readily. It is often worthwhile sampling from a wide variety of samples and perhaps pooling them to facilitate isolation, although this is also likely to increase the number on non-pathogenic bacterial colonies on the isolation plates.

4. Streak suspensions containing bacteria on the following media: a. Nutrient agar plate b. Nutrient dextrose agar plate c. Kings medium B plate d. 5% sucrose nutrient agar

The growth on these isolation plates may enable a presumptive diagnosis to be made See Table 1.

Isolation of Agrobacterium tumefaciens from galls

1. Wash the gall in tap water. 2. If the gall is old, rough or contaminated with soil, clean it thoroughly by

scrubbing gently, immerse in a suitable wetting agent (e.g. 0.1% Manoxol), surface sterilize by soaking in hypochlorite solution (0.5% available chlorine) for 10 minutes and rinse thoroughly in 3 changes of sterile water.

3. Slice pieces of young, fresh tissue from the gall surface. 4. Crush in a few drops of sterile water in a sterile Petri dish. 5. Leave the crushed gall for at least 20 minutes and preferably for several

hours to allow the bacteria to diffuse out of the tissues. 6. Streak the suspension on plates of nutrient dextrose agar.

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Table 1: Characteristics associated with colonies of pathogens on isolation plates 5% sucrose nutrient agar Kings B Nutrient dextrose agar Possible pathogen

Whitish, domed, smooth, mucoid (levan).

Whitish-grey, raised with diffusible yellowish green pigment that fluoresces blue-green under ultraviolet light1

Whitish-grey, raised, butyrous Green fluorescent pseudomonads of group Ia, IVa

Whitish-grey, raised As above As above Green fluorescent pseudomonads of group Ib, III, IVb

Convex, smooth, whitish to yellowish with greenish centre

As above As 5% SNA Green fluorescent pseudomonads of group II

Convex, smooth or wrinkled, whitish-green to yellowish-brown; diffusible or non-diffusible pigments may be produced

Whitish-grey but often producing pigments which do not fluoresce under ultraviolet light

Convex, smooth or wrinkled, whitish green, yellowish to brown, diffusible or non-diffusible pigments may be produced

Non-fluorescent pseudomonads

High convex to domed, smooth, creamy to yellowish

As 5% SNA. No diffusible pigment. High convex to domed smooth, mucoid, creamy to yellow; brown diffusible pigment produced rarely.

Xanthomonads

Raised, convex or domed, smooth mucoid (occasional levan production2) usually white to whitish-grey

Raised or convex, whitish-grey which do not fluoresce under ultraviolet light

Raised or convex, whitish-grey Soft rot erwinias

Convex, domed, smooth, mucoid, yellowish-orange

Raised or convex, smooth white- yellowish3

Raised, convex to domed, mucoid, yellowish-orange

Coryneform

1 Occasionally atypical, non-fluorescent forms of some pathogens of Group I are isolated; but they are common in P. syringae pv. morsprunorum, and are the rule in P. s. pv. persicae.

2 Erwinia amylovora produces whitish, domed, smooth, mucoid levan colonies.

3 Gram positive. Pink, blue or violet pigments are sometimes produced

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1.3. Confirmatory diagnosis

1.3.1 Xanthomonas After 3 days incubation at 25 C, Xanthomonas colonies isolated from affected tissue with typical disease symptoms should be high convex to domed, smooth, creamy to yellowish in colour on all media and with no diffusible pigment produced on Kings B agar. Further characteristics of Xanthomonas include:

1. Gram stain negative 2. Oxidase reaction negative 3. Catalase reaction positive 4. Inhibited by 0.1% tri-phenyl tetrazolium chloride (TTC)

1.3.2 Pseudomonas After 2-3 days incubation at 25 C, Pseudomonas syringae colonies isolated from affected tissue with typical disease symptoms should be whitish, domed, smooth and mucoid (levan positive) on SNA medium. P. syringae pv syringae isolates produce a diffusible yellowish-green diffusible pigment on Kings B medium which fluoresces blue green under ultraviolet light. P. syringae pv. morsprunorum isolates do not always produce this pigment. Both pathovars have the following properties common to LOPAT group Ia psedomonads:

1. Levan positive 2. Oxidase reaction negative 3. Pectate liquefaction negative 4. Arginine dehydrogenase negative 5. Tobacco hypersensitivity positive

Characteristics differentiating P. syringae pathovars syringe and morsprunorum are shown in Table 2.

Table 2: Distinguishing characters between Pseudomonas syringae pathovars morsprunorum and syringae. Character pv. morsprunorum pv. 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 Kings medium B

+/- -

Green-fluorescent diffusible pigment on Kings medium B

+/- +

1.3.3 Agrobacterium After 2-3 days incubation at 25 C, colonies of agrobacteria on all media should be neat, round, entire, smooth, domed and mucoid. Additional properties of agrobacteria include:

1. 3-keto lactose produced on lactose yeast extract plates after 2 days by biovar I strains but not by biovars II or III.

2. All agrobacteria are O/F test positive (oxidative)

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3. 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. Use large-leaved rapidly grown tobacco plants. White Burley is a commonly-

used cultivar. Where possible grow bacteria on nutrient agar. 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 0.3-0.4 at = 600 nm).

2. 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. It is necessary to use a narrow gage needle of approximately 0.6 mm external diameter. The diagonal of the needle aperture should be adjacent and parallel to the surface of the tissue.

3. 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. 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. Label the areas of the leaf injected.

4. 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. For Pseudomonas spp. incubate plants in a well-ventilated glasshouse at a temperature of less than 30 C. If growth cabinets are available incubate at 25 C and 85% relative humidity with a diurnal daylight regime of 16 hours.

6. Not all xanthomonads give a positive hypersensitive reaction in tobacco. It is therefore recommended to also use tomato or pepper seedlings. The reaction is generally weaker than for fluorescent pseudomonads. Tobacco plants should be held at 16 C for 4 days prior to inoculation and should be incubated at 33 C after inoculation.

7. A positive hypersensitive reaction is given by a rapid collapse and watersoaking of inoculated tissue, usually within 24 hours, but at least within 48 hours, followed by a dry, light brown necrosis of the watersoaked tissue within 3 days. Under ideal conditions, a positive result should be obtained within 24 hours. Yellowing or browning without collapse is not a positive reaction.

1.4. Host test for canker development Perform a host test using healthy susceptible species:

1. Stab soft stems of plants with a needle charged with culture (106-107 cfu per ml).

2. Symptoms appear more reliably and quickly if the site of inoculation is protected from desiccation by covering with polythene for 48 hours.

3. It may be possible to produce spreading lesions and dieback on young detached shoots standing in water.

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4. 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.

5. The canker phase of some diseases cannot be produced by inoculation at all times of the year.

1.5. Host tests for leaf and stem spot symptom development 1. Inoculate young plants covered with polythene sleeves 24 hours prior to

inoculation. Ensure that the polythene does not touch the plant. 2. Prepare a suitable inoculum by washing the growth from a nutrient agar or

other suitable agar medium in sterile water. Mix well and dilute in sterile water to obtain a population of approximately 105-106 cfu per ml.

3. 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.

4. Spray inoculum as a fine mist until the plant surface is wet, ensuring that upper and lower leaf surfaces are wetted, and cover immediately, or place in a humid chamber, to prevent drying out. Any low pressure, easily sterilizable sprayer can be used. A hypodermic syringe with a needle, the tip of which has been bent at right angles to the bevel by pressing it hard on a surface, is very convenient and produces a fine, easily directed, fan-shaped spray.

5. Incubate for 48 hours before removing the polythene sleeve or removing the plant from the humid chamber. Grow the plant on at approximately 20-25 C and 85% relative humidity with 12-16 hours light.

6. Observe from 3 days onwards for typical watersoaked lesions. These should become necrotic, rounded or angular, usually brown-black and usually with a watersoaked margin. Lesions are often elongated along stems.

7. If lenticular or stomatal infections do not occur, wound infection usually succeeds in producing symptoms but caution is then needed in interpreting the results. Repeat tests using different methods and conditions may be necessary to ensure accurate diagnosis.

8. 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.

1.6. Host test on immature walnut fruits for Xanthomonas arboricola pv. juglandis. 1. Swab immature healthy fruits with alcohol and wash in sterile water. 2. 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. 3. Incubate at 25 C in closed boxes lined with damp blotting paper. 4. Use a known reference culture of Xanthomonas arboricola pv. juglandis as a

positive control. 5. Use sterile water as a negative control.

1.7. Host test for Agrobacterium tumefaciens 1. Use the original host where this is possible. 2. If this is impractical, use several hosts. Tomato, marigold, sunflower,

Kalanchoe sp. and chrysanthemum are useful and easily cultivated. 3. Inoculate either by stabbing young stems or leaves with a needle through a

droplet of bacterial suspension (containing approximately 108 cfu per ml). 4. Galls usually develop within 2 weeks. Observe for gall symptoms for up to

28 days.

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2. Bacterial Taxonomy and Nomenclature


This section reviews the current taxonomy of the plant pathogenic bacteria, including the 3 pillars of taxonomy, classification, identification and nomenclature. Methods that can be used in identification include tried and tested traditional methods as well as modern molecular methods.

2.1 Classification Classification is the orderly arrangement of bacteria into groups. There is nothing inherently scientific about classification and different groups of scientists may classify the same or similar organisms differently. For example, clinical microbiologists are interested in the serotype and antimicrobial resistance patterns, whereas plant bacteriologists are concerned with pathovars, host specificity and virulence genes borne on mobilisable plasmids. Most taxonomists prefer a polyphasic classification based on a number of different attributes. Perhaps because these are time consuming and expensive, there is a trend towards phylogenetic classifications based on sequences of genes that mirror the evolution of the organisms. Initially this was largely based on the 16S rDNA but recently, other house keeping genes have been used, often in groups of up to seven, in a process referred to as multi locus sequence typing (MLST). Unfortunately, polyphasic and phylogenetic classifications do not necessarily coincide. The classification results in clusters of strains defined at various taxonomic levels including class, family, genus and species. Within plant pathogenic species there may be further grouping at subspecies, pathovar, biovar and race. The species is the gold standard here and is usually defined as a group of strains which share at least 70% DNA homology.

2.2 Identification Identification is the practical use of classification criteria to distinguish organisms from others, to verify the authenticity of a strain, or to isolate and identify the organism that causes a disease.

2.3 Nomenclature Nomenclature is the means by which the characteristics of a species or other taxon are defined, named and communicated. There is an internationally accepted code for nomenclature. Historically this has proved contentious for a number of published names, mostly through the upgrading and downgrading of taxonomic rank from and to pathovars and vice versa. Current approved names are regularly updated (4).

2.4 Diagnosis Diagnosis is the process of determining the cause of a disease. Although identification is an integral part of diagnosis, a diagnosis can usually be made with fewer tests than an identification of the isolate. There are 3 manuals that are invaluable to diagnosticians (1,2,3), although two are nomenclaturally out of date (1, 2). Between them they cover identification and diagnosis, although the approaches are somewhat different. Both processes have merit in the search for cost effective, easy to use, generic methods. For example, fatty acid profiling is a useful method that broadly differentiates bacteria at species level. 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.

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The addition of a host test raises the level from a presumptive diagnosis to a confirmed diagnosis. The former may be suited to a grower, the latter to a finding of statutory significance. There are several fairly recent key taxonomic revisions that hinder identification and sometimes diagnosis:

Some diseases e.g. bacterial spot of tomato and pepper with fairly similar symptoms can be caused by pathovars within several Xanthomonas species proposed as X. vesicatoria, X. axonopodis pv. vesicatoria, X. euvesicatoria and X. gardneri.

A fairly recent complication has been the concept of genomospecies, i.e. groups of strains which have less than 70% DNA homology but for which there are few tests to differentiate them. Within Pseudomonas syringae, most of these are not named but within what was Xanthomonas campestris, some 20 plus species have been classified and named. Identification at species level can thus be difficult and beyond the means of most diagnostic laboratories.

Another complication has been the mix of phenotytpic and phylogenetic classifications at different taxonomic levels. This is best exemplified in the Enterobacteriaceae, where the genus Erwinia has been divided on the basis of phylogenetic differences within the 16SrDNA genes sequences, whereas classification of species within them is still based largely on a polyphasic approach.

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. The ideal is a classification based on a polyphasic approach for which a single generic method gives a parallel classification. This method could then be used in identification. Genetic fingerprints, nutritional and protein profiles are very useful in deciding whether strains are the same or not. Fatty acid profiles give other information of taxonomic value eg chemotaxonomic information at genus level. As sequencing of DNA and whole genomes becomes more accessible, then phylogenetic methods such as sequencing of housekeeping genes is likely to become a more popular and available method. Some of the more recent evidence supports the use of 16S rDNA sequencing to differentiate genera, e.g. within the Enterobacteriaceae. And whereas 16S data cannot always reliably differentiate species, sequencing of other housekeeping genes usually can. However, it will be a long time before such methods are available to everyone and the more traditional methods must not be forgotten.

2.5 Gram Positive Bacteria The main genera, all in the Actinomycetales are Clavibacter, Curtobacterium, Leifsonia, Rathayibacter, Streptomyces, and Rhodococcus. . The first four of these are in the family Microbacteriaceae and are differentiated on the basis of 16S rDNA analysis. Clavibacter and Curtobacterium both have single pathogenic species comprising subspecies and pathovars respectively. PCR, genetic fingerprints and serological assays, all of which are also useful in diagnosis, most frequently differentiate these. Another problematic area is the identification of scab forming species within Streptomyces. It is likely that gene sequencing will help here.

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2.6 Gram Negative Bacteria, within the alpha Proteobacteria

The main genus is Agrobacterium. A recent proposal subsumes it within Rhizobium but there is resistance to this. Pathogenicity is governed by mobilisable plasmids. Six species are recognised but it is known that biovar 1 corresponding to A. radiobacter and A. tumefaciens, actually comprises many genomospecies at the 70% DNA homology level. Traditional tests (1, 2, 3) plus fatty acid profiling are useful both in diagnosis and differentiation. Within the beta Proteobacteria, the main genera are Acidovorax, Burkholderia and Ralstonia. For Acidovorax, which is not well studied, fatty acids are useful in genus determination. Genetic fingerprints such as repetitive sequence PCR perhaps best differentiate species and pathovars. Burkholderia is a complex genus for which the species are often difficult to identify. Again fatty acid analysis and genus specific PCR primers are useful for genus determination. Gene sequencing best differentiates species. Traditional tests are still useful for diagnosis (1, 2, 3). Ralstonia solanacearum is the sole pathogenic species. Identification and diagnosis at species level is straightforward and there are species-specific PCR primers as well as a range of other tests (1, 2, 3) Within the species, biovar, race and phylotype exist. The latter is the best but depends on gene sequencing. Biovar and race determination are widely described (1, 2, 3).

2.7 Gram Negative Bacteria, within the gamma Proteobacteria There are 3 main groups. The Enterobacteriaceae, once almost all incorporated within Erwinia, now has 8 genera proposed containing plant pathogenic species (Brenneria, Dickeya, Enterobacter, Erwinia, Pantoea, Pectobacterium, Samsonia, Serratia). Genus determination is largely based on 16SrDNA sequencing. The species within each can be differentiated by traditional tests (1, 2, 3) and by fatty acid analysis and repetitive sequence PCR. Diagnosis can be straightforward using traditional methods (2). Pseudomonas comprises many different species including genomospecies still classified within P. syringae. Most produce fluorescent pigments; fatty acid analysis is also useful for genus determination. Species and many pathovars within them can be differentiated by repetitive sequence PCR as well as traditional and host tests (1, 2, 3) Diagnosis is often straightforward (2). Xanthomonas now comprises some 20 species, many with pathovars. Genus determination can be achieved by fatty acid analysis but species determination is best achieved by gene sequencing e.g. gyrase B gene, which shows good correlation with DNA homology. Pathovar determination does not always rely on prior determination of species and traditional methods for identification and diagnosis (1, 2, 3) are still very useful.

References

1. Bradbury, J.F. (1986) Names of plant pathogenic bacteria. CABI, UK 2. Lelliott, R.A. and Stead, D.E. (1987) Methods for the diagnosis of bacterial

diseases of plants. Blackwell Scientific Publications, UK 3. Schaad, N.W., Jones, J.B and Chun, W (2001) Laboratory guide for identification

of plant pathogenic bacteria .3rd edition APS, USA 4. Young, J.M. et al. Names of Plant Pathogenic Bacteria

http://www.isppweb.org/names_bacterial.asp

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3. Primer design and Bioinformatics

Neil Boonham Central Science Laboratory, Sand Hutton, York, YO41 1LZ, UK [email protected]

3.1 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. In Europe you submit to EMBL; in Japan to the DNA databank of Japan; and in the US to the NCBI. The data is then shared among all these systems. There are a large, and ever growing, number of databases that you can search against. The databases can be searched in a number of ways; the most useful methods are searching for individual sequences of interest i.e. using a keyword, followed by a range of similarity based searches, i.e. searching a database for sequences similar to a query sequence.

3.1.1ENTREZ search ENTREZ search is a keyword search that allows you to search a range of databases including the nucleotide databases. This can be accessed in a number of ways; a simple ENTREZ search (http://www.ncbi.nlm.nih.gov/gquery/gquery.fcgi) searches all possible databases, reporting back all hits. For example; a search for Pytophthora ramorum:

Hyperlinks lead you through into the sequence accessions, papers, taxonomy etc. where the information can be either retrieved by cut and pasting or saving to files in a range of formats.

Sequences

Papers

Enter keyword here

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3.2 Retrieving sequence information Information can be removed individually following the hyperlinks, or in groups by (a) highlighting the individual items, then (b) selecting the format (e.g. Genbank, FASTA etc.) followed by (c) the form in which you want the information (e.g. save to file, text etc.) as follows. For sequence information the most useful formats are the summary, the Genbank file (the sequence accession) and FASTA format (for further analysis software).

(a) Highlight accessions (b) Select format (c) Select output

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3.3 BLAST searching The Basic Local Alignment Search Tool (BLAST) (http://www.ncbi.nlm.nih.gov/BLAST) for comparing gene and protein sequences against others in public databases now comes in several types including PSI-BLAST, PHI-BLAST, and BLAST 2 sequences. Specialized BLASTs are also available for human, microbial, malaria, and other genomes, as well as for vector contamination, immunoglobulins, and tentative human consensus sequences.

The simplest and most useful is a BLASTn or nucleotide-nucleotide BLAST. This returns information from the database about a query sequence, essentially allowing you to identify unknown sequences or confirm the identity of a known sequence. Remember the results only reflect what is present on the database, thus negative results (no significant matches may be difficult to interpret). Paste in your query sequence into the link, use the default setting to begin with for most searches (ignore the options section).

(a) Paste sequence here (b) Click on BLAST

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3.3.1 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). 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 (HSPs).

Sequence with highest score value

Alignment for the highest scoring pair (HSP)

Score value

Probability

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3.3.2 Interpreting the score values Complete explanation of the interpretation of the score values is beyond the scope of this protocol booklet, and can be found at (http://www.ncbi.nlm.nih.gov/BLAST/tutorial/Altschul-1.html). However it is important to understand the basics.

The score is the basis of the BLAST search. In an alignment of a pair of sequences each nucleotide is given a score depending on whether it matches or not. If there is no match the score is negative, and if there is a match the score is positive. For each region of alignment all the scores are added up, though the score can never go below zero. 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).

When you get a BLAST result you also get a probability value. 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. If you are comparing a short sequence it is more likely that a random sequence could give you the same result. 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.

A probability of 0 means that there is essential no chance your match was random. 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. This is not very likely, and so most hits returned with a score of 10-50 are probably real. In contrast a result of 0.1 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.

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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. It is also a useful tool to use prior to performing diagnostic PCR primers, 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). The most commonly used algorithm is CLUSTAL and can be performed at several web sites (e.g. http://www.ebi.ac.uk/clustalw/).

In both cases the input format is a list of sequences in FASTA format that can be collected following an ENTREZ keyword search, outputted to text and pasted into the alignment tool, accept the default settings to begin with.

3.4.1 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

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The alignments can be highlighted using a program such as BOXSHADE on http://www.ch.embnet.org/software/BOX_form.html. In this version an .ALN format file is produced by CLUSTAL W that is inputted (by cut and pasting) to give an output file in an .RTF format that can be viewed in Word.

Divergent nucleotide positions

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3.5 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. Although there are no universal rules for primer design and many people use different approaches several generalities can be borne in mind.

PCR primers are usually 18-25nt in length with a GC content of between 40-60 % and a melting temperature (Tm) range of between 55-65C. Clearly all of these factors are linked together, thus selecting the required Tm in primer design software should account for the other parameters.

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.

From a diagnostic point of view specificity is generally the most important aspect of primer design, i.e. targeting the correct region of sequence. With this in mind the primer can be conveniently split into 3 regions, when it comes to either exploiting sequence differences for discrimination or coping with sequence differences for universal amplification.

For discrimination the sequence differences should be in the 3 end of the primer, whilst differences in the 5 will have little effect on discrimination of closely related sequences. The converse is true for universal detection, 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.

5 3

Least Important < Less Important < Most Important

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3.6 Primer design practice

A large amount of software is available for primer design, both free and commercial, for general-purpose use many of the free packages are effective, for example (http://frodo.wi.mit.edu/cgi-bin/primer3/primer3_www.cgi). This package has a large number of preset defaults and options to customise the design to meet your requirements; the most useful are as follows.

The program outputs an optimal (most closely fits the program parameters) primer pair followed by some further possible designs. 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. Following PCR it is necessary to sequence the amplified products of the correct size, and confirm the identity by BLAST searching.

Indicate the position of the 3 end of the primer

Default parameters for primers these are ideal for most situations

Product size, small is best for diagnostic use

Paste sequence here

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4. Isolation of bacterial DNA

Jenny Tomlinson Central Science Laboratory, Sand Hutton, York, YO41 1LZ, UK [email protected]

4.1 DNA isolation using ChargeSwitch Technology (CST)

Several DNA extraction kits are available that work on the principle of binding DNA to magnetic particles, washing the particles to remove contaminants, and finally eluting the DNA from the particles. Many of these use magnetic silica beads, which bind DNA in the presence of chaotropic salts. In contrast, the ChargeSwitch gDNA Plant kit (Invitrogen) uses magnetic beads coated with a material with pH-dependent charge (Figure 1).

Figure 1. ChargeSwitch Technology for DNA extraction.

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, or can be automated, for example using the KingFisher mL (Thermo) (Figure 2).

Figure 2. Devices for manipulation of magnetic beads for DNA extraction: left, PickPen 8-M (BioNobile); right, KingFisher mL (Thermo) for automated handling.

+

Low pH High pH

ChargeSwitch magnetic beads have positive

charge at low pH: nucleic acid binds

Charge is neutralised >pH 8.5: DNA is instantly released

Charge Switch DNA extraction method

Lyse sample with SDS

Bind DNA to CST beads pH

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ChargeSwitch / KingFisher DNA extraction protocol

Reagents required: ChargeSwitch gDNA Plant kit (Invitrogen, cat # CS18000)

Safety Information: Please take care when using the following reagents: Lysis Buffer: contains Urea irritant SDS: sodium dodecyl sulfate irritant; harmful if swallowed Precipitation Buffer: contains potassium acetate and potassium chloride - irritant Detergent: Triton X-100 irritant; harmful if swallowed

You will also need: KingFisher mL, plus appropriate plastic ware (strips and combs) Microcentrifuge Pipettes (200l and 1000l) Sterile filter pipette tips Gloves Microfuge tubes Ice

Method

1. Chill the Precipitation Buffer on ice.

2. Add 900l Lysis Buffer to 100l sample.

3. Add 100l SDS, then incubate at room temperature for 5 minutes.

4. Add 400l cold Precipitation Buffer and vortex to mix.

5. Centrifuge at approx 12 000 x g for 5 minutes.

6. Transfer 1ml of clarified sample to a fresh microfuge tube, then add 40l CST Beads and 100l 10% Detergent and mix gently by pipetting.

Resuspend the CST Beads thoroughly before use.

7. 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: 200l Elution Buffer

8. Transfer the sample and beads to Well 1 of the KingFisher mL strip.

9. Insert new tip combs on the Kingfisher mL, and run programme gDNA (approximately 10 minutes).

10. Transfer the eluted DNA to a clean labelled microfuge tube.

Store the DNA extracts at -20C.

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5. PCR and real-time PCR protocols

Richard Thwaites Central Science Laboratory, Sand Hutton, York, YO41 1LZ, UK [email protected]

5.1 Conventional PCR PCR utilises short DNA sequences (primers) to amplify a specific target DNA sequence. Each primer hybridises to opposite strands of the target sequence and are orientated so that DNA synthesis, by Taq DNA polymerase, proceeds across the target sequence, effectively doubling the amount of the target DNA sequence. Since the extension products are also complementary to and capable of binding primers, the cycle can be repeated after a denaturation step. 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. In conventional PCR these copies are observed by running the reaction mix through an agarose gel. The gel is then stained with ethidium bromide. Ethidium bromide chelates to DNA and fluoresces under UV illumination (Fig. 1).

Fig. 1. Ethidium bromide stained gel (under UV illumination) showing PCR products of 224 bp.

5.2 Real time PCR Real-time (or TaqMan) PCR exploits the 5 nuclease activity of Taq DNA polymerase in conjunction with fluorogenic DNA probes. Each probe, designed to hybridise specifically to the target PCR product (between the two primers), is labelled with a fluorescent reporter dye and a quencher dye. During PCR amplification the probe is digested by Taq DNA polymerase, separating the dyes, and resulting in an increase in reporter fluorescence (Fig 2). 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.

Real-time analysis also facilitates quantification of the amount of sample DNA present in the reaction by ascertaining when (i.e. during which PCR cycle) fluorescence in a given reaction tube exceeds that of a threshold (Threshold Cycle (CT)). Lower CT values indicate higher amounts of target DNA (Fig 3). Comparison between reaction tubes and / or known standards can quantify the amount of DNA template present in a given tube.

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Although TaqMan chemistry has been described here, other real-time PCR chemistries exist. 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. As the PCR progresses increased amounts of DNA, and therefore fluorescence, are produced from positive reactions. This can be analysed in the same way as fluorescence emitted during TaqMAn real-time PCRs, though a post-PCR melt-curve analysis is required to ensure that the target PCR sequence has been amplified.

QR

Probe Cleavage

QR

PrimerProbe

Polymerisation and 5 Nuclease activity

Fluorescence absorbed

Primer

TAQ

TAQ

Fig. 2. Real-time PCR (TaqMan) chemistry

Ralstonia solanacearum : Enrichment Taqman PCR for 96, 72, 48, 24 and 0 hours growth in SMSB for 106

cells per mL of potato extract.

Fig. 3. Typical amplification plot showing increase in fluorescence from one target DNA molecule at different concentrations in the initial samples

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5.3 rep-PCR Characterisation of bacterial genomes has led to the recognition of repeated DNA sequences that are conserved within diverse bacteria, especially Gram-negative bacteria. Three DNA families have been recognised that are unrelated at the DNA level. These are referred to as repetitive extragenic palindromic (REP) sequences, enterobacterial repetitive intergenic consensus (ERIC) sequences and the BOX element.

Nucleotide sequence determination of these repetitive regions has enabled the design of PCR primers specific to each region. PCR with these primers thus give rise to amplification products that reflect each number and distribution of repetitive sequences.

This approach to genomic fingerprinting is referred to as rep-PCR and offers a highly sensitive level of analysis, suitable for species and in some cases pathovar / biovar identification.

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5.4 Protocols 5.4.1 Real-time PCR protocols for Xanthomonas arboricola (Weller et al. 2007,)

Primers and Probes

Primers Xaf pep-F 5' - GCG TGC CGC AGC CGC - 3' Xaf pep-R 5' - CCG GTG GGC TTG GCG CCG - 3'

Probes Xaf pep-P 5 - CCG GAA ACC GGC AAG AAG GCA - 3'

The real-time PCR with Xaf primers and probes detects strains of Xanthomonas arboricola pathovars pruni, corylina and fragariae.

PCR reaction mix

Reagent Quantity per reaction (l) Final concentration Sterile UPW 10.875 10x PCR buffer 2.5 1x MgCl2 (25 mM)) 3.5 3.5 mM d-nTP mix (2.5 mM) 2.0 200 M Forward Primer (10 pmol l-1) 0.75 300 nM Reverse Primer (10 pmol l-1) 0.75 300 nM Probe (5 pmol l-1) 0.5 100 nM Taq polymerase (5U/l) 0.125 0.63 U Sample volume 4.0

Total volume :

25.0

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).

Note: This programme has been optimised for use with the ABI 7700 and 7900 sequence detector TaqMan systems and the Cepheid Smartcycler system. Further optimisation of this programme may be required for use with other real-time PCR systems.

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5.4.2 rep-PCR for genomic fingerprinting Schaad (2001)

Primers REP1R-1 5 III ICG ICG ICA TCI GGC 3 REP2-1 5 ICG ICT TAT CIG GCC TAC 3

PCR reaction mix

Reagent Quantity per reaction Final concentration Sterile UPW 2.95 l 10x PCR buffer 2.5 l 1x MgCl2 (25mM) 6.7 l (6.7 mM MgCl2) d-nTP mix (25mM each) 1.25 l 1.25mM BSA (20 mg per ml) 0.2 l 4 g DMSO 2.5 l 10% Primer REP1R-1 (20M) 3.75 l 3 M Primer REP2-1 (20M) 3.75 l 3 M Taq polymerase (5U/l) 0.4 l 2.0 U Sample volume 1.0 l

Total volume :

25.0 l

REP-PCR reaction conditions

95OC - 2 min

94OC - 3 sec ) 92 OC - 30 sec ) 40OC - 60 sec ) x 35 cycles 65OC - 8 min )

65OC - 8 min 4OC - 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. Therefore care must be taken to follow the same protocol for each gel / run performed. Variation between gels will affect the quality of the analysis performed post-electrophoresis.

Prepare a 1.5% agarose gel (20 x 24 cm) in 1 x TAE buffer. Use a 20 or 30 tooth comb (1 mm).

On a strip of parafilm add 6-10 l amplified DNA mixed with 1.2-2 l of 6 x loading buffer (LB) and add the final mix into the wells on the gel. Leave three of the wells for the molecular weight marker - one at each end and one in the middle.

Run the gel at a constant voltage of 105V (approx 55 mA) per gel. Set the powerpack to run for 5-6 hours at room temp (or 70V/~23 mA for 16-18 hrs at 4 C).

40

After electrophoresis, switch off the power pack and remove the gel from the tank. Stain in ethidium bromide (final conc. 600ug/1000ml) CARE! - ETHIDIUM BROMIDE IS MUTAGENIC - wear latex disposable gloves. Stain for 30 mins and destain in water.

5.4.3 ERIC-PCR for genomic fingerprinting Schaad (2001)

Primers

ERIC1R 5 ATG TAA GCT CCT GGG GAT TCA C 3 ERIC2 5 AAG TAA GTG ACT GGG GTG AGC G 3

PCR reaction mix

Reagent Quantity per reaction Final concentration Sterile UPW 2.95 l 10x PCR buffer 2.5 l 1x MgCl2 (25mM) 6.7 l (6.7 mM MgCl2) d-nTP mix (25mM each) 1.25 l 1.25mM BSA (20 mg per ml) 0.2 l 4 g DMSO 2.5 l 10% Primer ERIC1R (20M) 3.75 l 3 M Primer ERIC2 3.75 Taq polymerase (5U/l) 0.4 l 2.0 U Sample volume 1.0 l

Total volume:

25.0 l

ERIC-PCR reaction conditions

95OC - 2 min




It is very important to standardise electrophoresis conditions as much as possible in order to minimise variation in band patterns between gels. Therefore care must be taken to follow the same protocol for each gel / run performed. Variation between gels will affect the quality of the analysis-performed post-electrophoresis.


On a strip of parafilm add 6-10 l amplified DNA mixed with 1.2-2 l of 6 x loading buffer (LB) and add the final mix into the wells on the gel. Leave three

41

of the wells for the molecular weight marker - one at each end and one in the middle.



5.4.4 BOX-PCR for genomic fingerprinting Schaad (2001)

Primers

BOXAIR 5 CTA CGG CAA GGC GAC GCT GAC G 3

PCR reaction mix

Reagent Quantity per reaction Final concentration Sterile UPW 6.7 l 10x PCR buffer 2.5 l 1x MgCl2 (25mM) 6.7 l (6.7 mM MgCl2) d-nTP mix (25mM each) 1.25 l 1.25mM BSA (20 mg per ml) 0.2 l 4 g DMSO 2.5 l 10% Primer BOXAIR (20M) 3.75 l 3 M Taq polymerase (5U/l) 0.4 l 2.0 U Sample volume 1.0 l

Total volume :

25.0 l

BOX-PCR reaction conditions

95OC - 2 min



42


It is very important to standardise electrophoresis conditions as much as possible in order to minimise variation in band patterns between gels. Therefore care must be taken to follow the same protocol for each gel / run performed. Variation between gels will affect the quality of the analysis-performed post-electrophoresis.


On a strip of parafilm add 6-10 l amplified DNA mixed with 1.2-2 l of 6 x loading buffer (LB) and add the final mix into the wells on the gel. Leave three of the wells for the molecular weight marker - one at each end and one in the middle.



43

References

Louws FJ, Fulbright DW, Stephens CT, DeBruijn FJ. 1994. Specific genomic fingerprints of phytopathogenic Xanthomonas and Pseudomonas pathovars and strains generated with repetitive sequences and PCR. Applied and Environmental Microbiology 60: 2286-2295.

Pastrik, K. H., Elphinstone, J.G. and Pukall, R. 2002. Sequence analysis and detection of Ralstonia solanacearum by multiplex PCR amplification of 16S-23S ribosomal intergenic spacer region with internal positive control. European Journal of Plant Pathology 108, 831-842

Pastrik, K-H. 2000. Detection of Clavibacter michiganensis subsp. sepedonicus in potato tubers by multiplex PCR with coamplification of host DNA. European Journal of Plant Pathology 106; 155165.

Schaad, W., Berthier-Schaad, Y., Sechler, A. and Knorr, D. 1999. Detection of Clavibacter michiganensis subsp. sepedonicus in potato tubers by BIO-PCR and an automated real-time fluorescence detection system. Plant Disease 83; 1095-1100.

Versolavic J, Koeuth T, Lupski JR. 1991. Distribution of repetitive DNA sequences in eubacteria and application to fingerprinting of bacterial genomes. Nucleic Acids Research 24: 6823-6831.

Weller, S.A., Elphinstone, J.G., Smith, N., Stead, D.E. and Boonham, N. 2000. Detection of Ralstonia solanacearum strains using an automated and quantitative flourogenic 5 nuclease TaqMan assay. Applied and Environmental Microbiology 66; 2853-2858.

Weller, S.A., Beresford-Jones, N.J., Hall, J., Thwaites, R., Parkinson, N., Elphinstone, J.G. 2007. Detection of Xanthomonas fragariae and presumptive detection of Xanthomonas arboricola pv. fragariae, from strawberry leaves, by real-time PCR. Journal of Microbiological Methods 70; 379383.

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APPENDIX

1) 50 x TAE buffer

242 g Trizma base (Sigma T-840) 18.6 g diSodium EDTA 57.1 ml glacial acetic acid

Make up to 1 litre with distilled water. Check pH is 8.0

2) 5 x loading buffer

5 to 8 mg bromophenol blue in 10 mls 50% glycerol.

BECAUSE GLYCEROL IS VERY VISCOUS, 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.

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6. Minimum requirements for diagnosis


6.1 Diagnosis or Identification: Detection or Diagnosis

These terms are often used wrongly! If they are fully understood and used correctly, much time and effort can be saved in determining the cause of a plant disease.

Identification is the process of allocating a name to an unknown bacterium. The starting point is having a pure culture.

Diagnosis is the process of determining the causal agent of a disease. The diagnosis may be presumptive i.e. with around 90% chance of being correct, or confirmatory i.e. 100% chance of being correct. A Presumptive diagnosis usually comprises isolation plus preliminary identification of some sort. A confirmatory diagnosis requires a host test (to complete Kochs postulates) although, increasingly, molecular methods are getting close to replacing the need for a host test in some situations.

Detection is the process of demonstrating the presence of a specific bacterium in a sample. Detection is often based on a single test/method e.g. IF or PCR. Detection can give a presumptive diagnosis

The approach to these issues can be seen in the various books written on diagnosis and identification. These include:

Laboratory guide for identification of plant pathogenic bacteria (3rd ed.) by Schaad et al. (2001). This is a book primarily about identification.

Methods for the diagnosis of bacterial diseases of plants by Lelliott &Stead 1987. This is a book primarily about diagnosing the diseases.

The time taken to achieve a diagnosis is often critical. Rapid diagnosis is needed to allow appropriate control. The more diagnoses that can be completed by a diagnostician in a day, the more cost effective the service becomes. The diagnostic requirements or level of diagnosis needed will depend on the value of the diseased crop or consignment. There may be less value in confirming the cause of a single leaf spot in a field than a consignment of high value, imported seed potatoes with high disease incidence.

There is a common tendency to isolate the bacterium and then start the process of identification, which will almost certainly take more time than is necessary. The most important pieces of diagnostic information are the symptoms themselves and with an experienced eye, a presumptive diagnosis may be made at this point. It is highly cost effective to know what bacteria/other pathogens can cause a particular type of symptom in a host. There are usually not many that can. 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, another process open to unnecessary complication and creation of more work than is necessary. As a visual infection develops, the symptomatic tissue may contain the pathogen but often in a decline

46

phase of growth or in competition with secondary invaders. The leading edge of the symptomatic tissue, 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. Your skill at selecting the correct leading edge is defined usually by the purity of the isolation you make. Successful pathogen isolation is often indicated by a pure culture of a bacterium with high colony numbers on a streak plate. 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, your presumptive diagnosis may be made here. Again, this will depend on your experience with the disease and the likely pathogens. In these days of exciting molecular methods we often ignore such valuable diagnostic information as pigmentation (soluble or insoluble), colony size, shape, elevation, edge, surface texture. If your plate is very mixed, it may be better to discard it and start again, than to attempt to identify all colony types in the hope that you may have the pathogen somewhere on the plate.

Assuming you have not been able to make a presumptive diagnosis at this stage, then the process of further characterisation or identification starts. Your lab may have decided to introduce a single standardised identification system such as fatty acid analysis or gene sequence based identification. Even if it has, 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. A simple Gram stain will usually differentiate Gram positive and negative bacteria. Many of the traditional tests, so rarely performed these days, are rapid, cheap and simple and often allow cost effective elimination of the contenders. These include oxidase test, pectolytic ability, arginine dihydrolase activity, and ability to grow at selected temperatures, pH or salt concentrations. However it is important to understand the significance of the tests applied. The presence of cytochrome c oxidase as in the oxidase test has greater evolutionary significance than the abilty to utilise mannose rather than galactose. Whereas all strains of a species are likely to be either oxidase positive or negative, 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, that rarely vary within strains of a given species. The LOPAT class gives perhaps as much diagnostic information as any modern method. For example, if you are presented with a bean leaf showing typical yellow haloes you may decide that only P. savastanoi pv. phaseolicola can cause those symptoms and make a presumptive diagnosis. If you are not entirely happy with this, and you go further and get a pure culture of a blue fluorescent bacterium on Kings B medium, you may also decide to make a presumptive diagnosis. If you are still not convinced, then a negative oxidase test could clinch it or at most a LOPAT group 1, which additionally indicates a hypersensitive response in tobacco and thus a pathogen. 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, would a confirmatory host test likely to be required. In this case, it matters not whether the bacterium belongs to the species P. savastanoi or to P. syringae, but this is not always the case. Until recently there was no cost effective and relatively simple way of allocating xanthomonads to species. Species identification was based on the inference that pv xxxx belonged to X. arboricola whereas pv. yyyyy belonged to X. hortorum. Thankfully it seems that gene sequencing can now give classifications that agree with those based on complex DNA homology.

Of the major diseases covered in this COST action, most can be effectively diagnosed presumptively based on symptoms, a pure culture with a typical morphology and perhaps one or two other key tests. The main problems may arise

47

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. It is then that the molecular methods such as gene sequencing and repetitive sequence PCR come into their own.

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7. Fatty acid profiling

David E. Stead and John Heeney Central Science Laboratory, Sand Hutton, York, YO41 1LZ, UK [email protected] and [email protected]

7.1 Summary

Fatty acid profiles are well established for classification and identification of plant-associated bacteria. Profiles are based on standardised culture, simple chemical extraction and gas chromatographic separation of fatty acid methyl esters (FAMEs). FAMEs are identified and quantified to give a profile for each strain. Over 100 fatty acids of between 8 and 20 carbon atoms length, belonging to several classes, occur in bacteria. Most bacteria have between 5 and 30 of these acids. The types occurring are usually indicative of the class, family and genus. Many Gram-negative genera have unique series of hydroxy acids. Gram-positive genera usually lack hydroxy acids but are rich in branched acids. Acidovorax, Agrobacterium, Burkholderia, Clavibacter, Pseudomonas, Ralstonia, Rhodococcus, Xanthomonas and Xylophilus all have characteristic profiles. Fatty acid profiles do not tend to support genomospecies within Pseudomonas syringae but for most other species correspond well with DNA and rRNA homology.

The relative amounts of individual fatty acids present often allow accurate differentiation at species, subspecies and sometimes biovar and pathovar level. Pathovars in some species, e.g. Pseudomonas syringae, are often not well differentiated. Automated identification is facilitated by comparison with libraries (databases) of profiles produced under standard conditions. These can be self-generated or purchased. Accuracy of identification can be excellent

7.2 Introduction

One method of identifying bacteria, which has proven to be an important and cost effective tool, is fatty acid profiling. This method is widely regarded as one of the best methods for rapid, accurate and inexpensive identification of bacteria. Bacteria alter their lipid fatty acid composition to maintain membrane fluidity under varying environmental conditions. It is essential therefore to maintain strictly controlled growth conditions to ensure consistent and reliable results. Bacterial membranes contain lipids in concentrations of 0.2 - 50% (usually 5 - 10% dry weight) which can be extracted, converted to fatty acid methyl esters (FAMEs), and analysed by gas chromatography quantitatively and qualitatively.

The type and percentage of individual fatty acids present in bacteria not only varies from genus to genus, and species to species, but in some cases it is also unique at biovar or pathovar level. This system allows for identification of many, but not all bacteria. It uses the Sherlock Microbial Identification System which is used worldwide in clinical and environmental laboratories to identify over 1,500 aerobic and anaerobic bacterial species. It also uses the NCPPB3 library; 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. In order to analyse and identify an isolate of a bacterium, we need to obtain sufficient numbers of cells (approximately 40 mg). To meet this requirement we culture the cells under strict conditions and then harvest them for analysis purposes. Once the

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isolate has been cultured and harvested, the fatty acids can be extracted, converted to fatty acid methyl esters (FAMEs), and analysed.

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. Reference can also be made to a commercially available library (TSBA6.0), although it should be noted that conditions for culturing are not exactly the same as those used for the NCPPB reference organisms. The TSBA6.0 commercial library is based on cells cultured for 24 hours. For the NCPPB library, cells are cultured for 24 hours or 48 hours depending on species and genus.

Fatty acid profiling relies on separating the fatty acids according to their size and conformation. This procedure uses a Hewlet Packard HP6890 series Gas Chromatograph. The Hewlet Packard HP6890 series GC is controlled by HP Chemstation software. This, in turn, interfaces with Midi Sherlock software. This software enables comparison with a commercially available library (TSBA6) and an in-house generated library (NCPPB3). This latter library is based on the cultures held in the National Collection of Plant Pathogenic Bacteria (NCPPB), which represents nearly all known plant pathogenic taxa.

The GC uses a commercially available calibration standard, which is analysed twice prior to each set of runs and is also analysed once at set intervals during the run sequence. If calibration fails, the run automatically stops. Samples are analysed, the resulting profiles are compared to the libraries and an analysis report is automatically generated.

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. A comparison is made to the library entries and a profile is produced complete with a similarity index, and histoplots showing the values of the sample compared to the nearest library entries. When used in conjunction with selected traditional and more recently developed diagnostic methods it can be a very informative diagnostic tool. An experienced operator can prepare many samples in a day, leave the machine running overnight and obtain results the following day.

7.3 FAMEs

FAMEs are fatty acid methyl esters of the fatty acids released from cellular lipids. They are converted to FAMES to make them more volatile for gas chromatographic analysis. Most bacteria contain between 5 and 30 fatty acids, which are generally 8-20 carbon molecules in length. There are several major classes of fatty acids in bacteria; primarily straight chain saturated (16:0), straight chain mono-unsaturated (16:1 w7cis), cyclopropanes (17:0 cycopropane), iso-branched (15:0 iso), anteiso-branched (17:0 anteiso), 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. In contrast, Gram positives have branched acids and rarely have hydroxy acids. Some genera have rare acids e.g. the presence of 13: 0 iso 3OH is virtually exclusive to Xanthomonas. Table 1 shows the diagnostic patterns of hydroxy acids for various genera of Proteobacteria (Gram negatives)

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7.4 Methods

In order to obtain reproducible results, bacteria must be grown under uniform conditions of time, temperature and nutrients. We use 24 hour growth on Trypticase Soy Agar (TSA) at 28 C and harvest c. 40 mg growth from confluent growth avoiding the first (wash-out) and last (individual colonies) streaks.

The extraction procedure has several steps: 1. Cells are harvested and boiled in sodium hydroxide and methanol to saponify

the lipids in the cells 2. The resulting fatty acids are converted into methyl esters by heating in

hydrochloric acid and methanol 3. 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. The organic phase is washed with sodium hydroxide, removed and injected

into the gas chromatograph for analysis 5. For fastidious pathogens incubation time can be extended but must be the

same as used in the library development.

The FAMES are then identified according to their equivalent chain length and quantified. The profile is printed off listing all the named peaks and their peak areas. This profile is then compared with the libraries and matches given as a similarity index. Similarity indices of 0.7 or above are generally accurate at species level.

As well as the match, the chemotaxonomic information provided can be extremely useful. Gram-negative and Gram-positive bacteria have different profile types with key types of fatty acid present or absent. For Proteobacteria (Gram negatives), the presence/absence of hydroxy acids can be diagnostic at genus level. 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, though this is a generalisation (see Table 1).

It must be remembered that, if using a library, accuracy of identification can only be as good as the quality and content of the library. If the species you have is not in the library it cannot be identified. 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 less than 0.5 even though they may be first choice in the library are rarely correct identifications.

Thus the method cannot replace the diagnostician but together with the other diagnostic information, fatty acid analysis can be a very cost effective and useful diagnostic method. Although the initial costs of equipment and software is high, running costs are low.

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Table 1. Characterisation of Proteobacteria genera by presence/absence of specific hydroxy acids

FATTY ACID 10:0 3OH

12::0 2OH

12:0 3OH

14:0 3OH

16:0 2OH

16:0 3OH

16:1 2OH

18:1 2OH

11:0 iso3OH

13:0 iso3OH

Alphaprot

Agrobacterium + + (+) Betaprot

Acidovorax +

Burkholderia + + + + +

Ralstonia + + + +

Gammaprot

Erwinia +

Pseudomonas + + + (+) Xanthomonas + + +

.

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8. Identification of bacteria by partial gene sequencing

Neil Parkinson Central Science Laboratory, Sand Hutton, York, YO41 1LZ, UK [email protected]

8.1 Introduction

Microbial identification based on partial gene sequence data is now a commonly used procedure. 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. Comparisons of the sequence between different species suggest the degree to which they are related to each other; a relatively greater or lesser difference between two species suggests a relatively earlier or later time in which they shared a common ancestor. For closely related organisms, 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.

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.

These methods offer increasingly higher levels of discrimination between closely related bacteria.

The methods involve 3 stages: PCR amplification of the target sequences Purification and analysis of the amplified DNA Sequencing of the amplified DNA

8.2 Procedures

8.2.1 Preparation of bacterial cultures for sequencing

1. Suspend isolated bacterial colonies removed from pure cultures on agar plates in molecular grade water.

2. Heat 100 of suspension at 96 C for 4 min (Gram ve) or 15 min (Gram +ve). 3. Dilute in a further 900 l of molecular grade water. 4. Store at 20 C until required. 5. Use 2 l of suspension as DNA template per PCR reaction.

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PCR amplification of the target sequences

Perform PCR reactions as required according to the following reaction conditions:

8.2.2 16S rRNA partial gene sequence (Weisburg et al., 1991)

1. For each reaction prepare the following master mix ufp1 2.0 l urp1 2.0 l Molecular grade H2O 19.0 l Fermentas 2X PCR Mastermix 25.0 l

Primers: ufp1 AGT TTG ATC CTG GCT CAG (18 bp) urp1 GGT TAC CTT GTT ACG ACT T (19 bp)

2. Add 48 l master mix per PCR tube. 3. Add 2.0 l DNA template into each tube. 4. Run PCR programme as follows:

One cycle of : 95 C for 9 min Then 35 cycles of: 95 C for 1 min 55 C for 2 min 72 C for 1 min One cycle of: 72 C for 7 min Hold at: 4 C

(Ensure that PCR machine is programmed for 50 l reactions)

8.2.3 16-23S rRNA intergenic spacer region partial sequence (Prez-Luz et al., 2004)

1. For each reaction prepare the following master mix: 16s14f 2.0 l 23sor 2.0 l Mol.grade H2O 19.0 l Fermentas 2X PCR Mastermix 25.0 l

Primers: 16s14f CTT GTA CAC ACC GCC CGT C (19 bp) 23sor TGC CAG GGC ATC CAC CGT G (19 bp)


One cycle of : 94 C for 2.5 min Then 35 cycles of: 94 C for 30 sec 60 C for 30 sec 72 C for 1 min One cycle of: 72 C for 5 min Hold at: 4 C


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8.2.4 Pseudomonas gyrB partial sequence (Sarkar et al., 2000)

1. For each reaction prepare the following master mix: Forward primer 0.75 l Reverse primer 0.75 l Molecular grade H2O 10.0 l Fermentas 2X PCR Mastermix 12.5 l

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)


One cycle of : 94 C for 5 min Then 30 cycles of: 94 C for 2 min 63 C for 1 min 72 C for 1 min One cycle of: 72 C for 7 min Hold at: 4 C


8.2.5 Xanthomonas gyrB partial sequence (Parkinson et al., 2007)

1. For each reaction prepare the following master mix: XgyrPCR2F 0.75 l X.gyr.rsp1 0.75 l 10x Long PCR buffer + Mg 2.5 l dNTP mix (2.5mM each) 2.0 l Long PCR Enzyme Mix (Taq) 0.125 l Molecular grade H2O 17.875 l

Primers: XgyrPCR2F AAG CAG GGC AAG AGC GAG CTG TA (23 bp) Xgyr.rsp1 CAA GGT GCT GAA GAT CTG GTC (21 bp)

2. Add 24 l master mix per PCR tube 3. Add 1.0 l DNA template into each tube 4. Run PCR programme as follows:

One cycle of : 94 C for 2.5 min Then 34 cycles of: 94 C for 30 sec 50 C for 45 sec 68 C for 1 min One cycle of: 68 C for 7 min Hold at: 15 C


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8.2.6 Gel electrophoresis

1. Prepare 1,5 % agarose gel in 1X TBE buffer. 2. Mix 5 l of each PCR product with 5 l water and 2 l 6X loading dye on

parafilm and load into wells in the gel. 3. Add 4 l of 100 bp DNA ladder into the last well. 4. Run for about 2h at 104 V. 5. Stain the gel in ethidium bromide (600 g per L) for 30 min and de-stain in

water 6. View bands under UV transillumination

8.2.7 Purification of PCR product

If the PCR shows a single clear band the PCR product should be purified e.g. using the Wizard SV gel and PCR clean-up system (Promega A9280) as follows:

1. Add an equal volume of Membrane Binding solution to the remaining PCR product (45 l for 50 l PCR or 20l for 25l PCR).

2. Place in an SV minicolumn in a collection tube and incubate for 1 min at ambient temperature.

3. Centrifuge at maximum speed in a microcentrifuge for 1 min. 4. Discard liquid in the collection tube. 5. Add 700 l Membrane Wash solution (mixed with ethanol according to the

instructions with the Promega kit). 6. Centrifuge at maximum speed in a microcentrifuge for 1 min. 7. Discard liquid in the collection tube. 8. Add 500 ml of Membrane Wash solution (mixed with ethanol according to the

instructions with the Promega kit). 9. Centrifuge at maximum speed in a microcentrifug

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