Bacterial Wilt Disease

Ralstonia solanacearum

Standard Operating Procedure for Use in Diagnostic Laboratories

Version: EA-SOP- RS1 May 2014

CONTRIBUTORS

Name Specialization Institution Contact Dr. Z.M. Kinyua Plant Kenya Agricultural PO Box 14733-00800, pathologist Research Institute NAIROBI, KENYA [email protected] Prof. S.A. Miller Plant The Ohio State University Ohio Agricultural pathologist Research and Development Center Dept. Plant Pathology Ms. Ashlina Consulting The Ohio State University 1680 Madison Ave. Chin plant Wooster, OH pathologist USA 44691 [email protected] Mr. Nagendra Graduate The Ohio State University [email protected] Subedi research [email protected] assistant

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TABLE OF CONTENTS

PART 1. BACKGROUND ...... 1 1.1. Introduction ...... 1 1.2. Importance of bacterial wilt ...... 1 1.3. Symptoms/indications of bacterial wilt ...... 2 1.4. Pathogen description ...... 3 1.5. Transmission/spread of bacterial wilt pathogen ...... 3 PART 2. DIAGNOSIS / IDENTIFICATION ...... 4 2.1. Sample receipt and examination ...... 4 2.2. Sample storage ...... 4 2.3. Sample analysis ...... 4 2.3.1. Initial Screening ...... 4 2.3.1.1. Indicative symptoms and signs in infected plant materials ...... 5

2.3.1.2. Isolating Ralstonia solanacearum from infected plant materials ...... 6

2.3.2. Advanced Screening in Specialized Laboratories ...... 7 2.3.2.1. Classical analysis for biovar determination ...... 7

2.4. Confirmation ...... 13 2.5. Communication ...... 14 2.6. Sample Destruction ...... 14 Appendix 1: Media for isolation and cultivation of Ralstonia solanacearum ...... 15 Appendix 2: Ralstonia biovar testing ...... 17 Appendix 3: Reagents for DNA analysis ...... 19 Appendix 4: Bibliography ...... 20

ii PART 1. BACKGROUND

1.1. Introduction

Bacterial wilt of solanaceous plants, which is caused by the bacterium known as Ralstonia solanacearum (formerly Pseudomonas solanacearum), has been and continues to be a devastating disease in many countries. The bacterium, which is often endemic in the soil, penetrates the plant through the root system and eventually causes irreversible wilting and death. The disease has caused serious upsets to many farmers, particularly those who have been venturing into greenhouse production of crops that are susceptible to the bacterium. It is noteworthy that in the last 30 years, glasshouse cultivation of tomato has been widely adopted in mild and warm climate regions (Albajes et al., 1999). With this growing industry, where high investments are a reality, incidences of bacterial wilt are particularly devastating.

The bacterium affects tomato, potato, eggplants, capsicums and other cultivated and wild plants, including weeds such as black nightshade. The disease is also referred to as brown rot in potato or southern wilt in geranium.

In order to prevent initial infection or manage already infected crop fields, it is important to have clear protocols for diagnosis of the disease as well as for detection of the causal agent in plants, soil, water and other materials. This document is useful in the diagnostics of bacterial wilt and its causal agent in tomato and, by extension, potato, capsicum, eggplant and other similarly herbaceous plants. 1.2. Importance of bacterial wilt

Ralstonia solanacearum has a wide host range, which makes it difficult to have a generalized estimate of the economic losses caused by the bacterial wilt disease. Direct yield losses vary widely according to host, cultivar, climate, soil type, cropping practices and pathogen strain. Therefore, the level of damage is commonly expressed on a crop-by-crop basis, and can range from minimal crop loss to very high economic damage. Some examples are outlined below:

Kelman (1953) cites a serious case by quoting destruction of many potato and tomato fields in the southeastern United States, where the disease put an end to commercial tomato growing in certain regions such as southern Mississippi, southern Alabama and parts of Florida. During the period from 1920 to 1940, the sociological and economic impact of the disease in Granville County was felt by hundreds of farm families. Due to loss of income from their main cash crop, many farmers were forced to sell their farms and leave Granville County in search of new employment.

In India, a yield loss study with one cultivar of tomato showed 10-100% mortality of plants and 0-91% yield loss (Elphinstone, 2005). In Uganda, a survey of 17 districts showed that 88% of tomato farms were affected by bacterial wilt (Katafiire et al., 2005).

Ralstonia solanacearum causes brown rot in potatoes, which is known to cause yield losses in the field through plant death. Yield losses continue during storage and transit due to rotting and decay leading to even more revenue loss. The disease has been estimated to affect three million farm families, which accounts for about 1.5

1 million Ha) in around 80 countries. Currently, global damages exceed $950 million annually (Elphinstone, 2005). In Bolivia, potato yield loss at harvest ranged from 30- 90% and losses during storage were as high as 98% (Coelho and Nutter, 2005). Reports from Bangladesh quote some regions as having more than 30% of potato crops affected by R. solanacearum, with over 14% reduction in yield (Elphinstone, 2005). In Nepal, tuber rotting occurred in an average of 10% of stored potato, with a maximum of 50%. Crop losses in small farms in the Nepalese hills were up to 100%, mainly due to poor cultural practices, such as keeping seed from infected crop (Elphinstone, 2005). Potato yield losses in Uganda are estimated at 30% (Alacho and Akimanzi, 1993), with more severe losses being 100% (Opio, 1988; Kakuhenzire et al., 1993). In the South-western Uganda districts, 26% yield loss has also been reported (Lemaga et al., 1997). Various reports from Kenya have indicated that there is an increase in the incidence of brown rot of potato due to the spread and build-up of the disease in the majority of the potato growing zones (Ajanga, 1993; Barton et al., 1997; Ateka et al., 2001; Kinyua et al., 2001).

In addition to causing yield losses in field crops, management efforts for prevention, eradication, and general control of R. solanacearum are extremely costly, which contribute heavily to economic losses. The bacterium has also raised serious concerns in the floriculture industry, especially those producing geraniums (Daughtrey 2003, Kim et al 2003; Williamson et al 2002; Kim, 2002). Under such circumstances, trade in suspect planting materials and cut flowers has sometimes been subjected to trade restrictions.

1.3. Symptoms/indications of bacterial wilt

R. solanacearum primarily enters plants through natural openings or from wounds, particularly in the roots. Natural openings are commonly formed by lateral root emergence, while wounds are a result of root damage caused by soilborne organisms (e.g., the root-knot nematode), transplanting, cultivation or insects. The bacterium can also enter plants through stem injuries, although less common. Once the bacterium enters the plant, it spreads upward via the xylem and colonizes in the vascular bundles. This leads to a condition in which infected plants start wilting irreversibly.

Disease distribution may range from a few scattered plants or loci of infection in fields where low or erratic natural infestations occur to larger areas of wilting and dead plants in a field (Kelman and Sequeira, 1965). Under natural conditions, the initial symptom in mature plants is wilting of upper leaves during hot days followed by recovery throughout the evening and early hours of the morning. The wilted leaves maintain their green color as the disease progresses. Under hot and humid conditions, complete wilting occurs and eventually the plant dies. Occasionally, one-sided wilting is noticed whereby only some branches/shoots in a plant are seen to exhibit wilting (Agrios, 2005). Massive invasion of the cortex might result in the appearance of water-soaked lesions on the external surface of infected stems. If an infected stem is cut crosswise, tiny drops of dirty white or yellowish viscous ooze exude from several vascular bundles (Champoiseau et al., 2009b). There may also be discoloration of the vascular system, expressed in form of pale yellow to dark brown colour (Gota, 1992).

In addition to the above symptoms, infected potato plants may produce tubers that exhibit browning of the vascular ring and/or bacterial ooze when cut transversely. Soil may also be

2 seen to adhere to the eyes of such tubers as a result of the soil particles sticking on the bacterial ooze.

A plant infected with R. solanacearum may express all or none of the symptoms outlined above, even under environmental conditions that are ideal for the pathogen. If symptoms are not evident on an infected susceptible host, the condition is known as latency.

1.4. Pathogen description

R. solanacearum is gram-negative, rod-shaped bacterium measuring 0.5-0.7 × 1.5-2.0 µm in size. It grows well at 28 to 32°C strictly in aerobic conditions (Hayward, 1991; Schaad et al. 2001). Individual colonies of normal or virulent isolates are usually visible after 36 to 48 hours, appearing as opaque white or cream-colored colonies that are irregularly shaped and highly fluidal on a cultivation medium such as casamino acid-peptone-glucose-agar (CPG). On tetrazolium chloride (TZC) medium, these colonies will be white with pink centres (Kelman, 1954). Mutant or non-virulent type colonies of R. solanacearum are uniformly round and dark red, smaller in size, and butyrous or dry on TZC.

Strains of R. solanacearum have previously been grouped into five races based on susceptible host plants and biovar classification, which is determined by utilization of a panel of five to eight carbohydrate substrates (Table 1; Denny and Hayward, 2001). Since host ranges of strains are broad and often overlap and tests to define races are cumbersome, it is preferable to designate isolate biovars and determine their phylotype. Phylotyping is based on DNA sequence analysis, which divides the R. solanacearum species complex into four phylotypes. Phylotype I strains originated in Asia; phylotype II strains originated in the Americas; phylotype III strains in Africa; and phylotype IV strains in Indonesia. Phylotypes can further be subdivided into sequevars based on the sequence of the endoglucanase (egl) gene (Fegan and Prior, 2005; Prior and Fegan, 2005).

Table 1. Races and biovars of Ralstonia solanacearum Race Host Range Geographic distribution Biovar 1 Wide Asia, Australia, Americas 1, 3, 4 Banana Caribbean, Brazil 2 Other Musa spp. Philippines 1 Potato, some other 3 Solanaceae, Geranium; Worldwide except US and 2 plus a few other species Canada 4 Ginger Asia 3, 4 5 Mulberry China 5 (Source: Daughtrey, 2003) 1.5. Transmission/spread of bacterial wilt pathogen

Ralstonia solacearum survives in infected plants, plant debris, soil, water, weed hosts, seeds and vegetative propagation material. The pathogen spreads through movement of soil, irrigation with contaminated water, use of infected vegetative planting material, and mechanically via workers, tools, insects and equipment.

3 PART 2. DIAGNOSIS / IDENTIFICATION

2.1. Sample receipt and examination

Plants exhibiting symptoms of bacterial wilt or suspected to be infected by R. solanacearum may be received from farmers, agricultural extension officers, researchers, educators, regulatory service agencies, or private services providers. Other material may be water used for irrigation, or soil or other growth medium. Sampled materials should ideally reach the laboratory (analytical facility) within 24 hours from the time sampling takes place.

Upon receipt, the details of the material should be registered on sample record file/form prior to being subjected to diagnostic analysis. Analysis of received samples should commence as soon as possible in order to obtain results that represent the actual situation and to facilitate timely advice to the client from whom the samples are taken.

In case the sample requires to be despatched to other laboratories for analysis, care should be taken to ensure that the sample does not spoil or is not released into the environment to prevent contamination or cause unintended infections. This demands proper packaging/wrapping to ensure appropriate sealing. The material should also not be exposed to high temperatures, which can cause sample spoilage. A sample to be despatched should be labelled properly to show the type of material, source/location of sampling, name of sampler, date sampled and analysis required.

2.2. Sample storage

While analysis should be initiated as soon as a sample is received, it may be necessary to store the sample or parts of sample for subsequent use/reference. It is advisable to keep such sample under refrigeration or in a cool room to avoid spoilage.

Pure cultures of R. solanacearum resulting from isolation procedures can be stored for many years at room temperature in sterilized tap, distilled or deionized water, or at -80°C in liquid culture broth amended to 40% glycerol. R. solanacearum easily loses virulence if repeatedly transferred on agar plates and loses viability if plates are stored at 4°C. Cultures can become non-culturable, although viable, if exposed to very low temperatures (van Elsas et al., 2001).

2.3. Sample analysis

2.3.1. Initial Screening

Initial screening involves tests such as bacterial streaming, plating on a semi-selective medium, such as an improved semi-selective medium from South Africa (SMSA; see Appendix I) developed by Engelbrecht (1994) and further modified by Elphinstone et al. (1996, 1998), immunodiagnostic assays using species-specific antibodies, polymerase chain reaction (PCR) with species-specific primers, and pathogenicity tests using susceptible hosts such as tomato seedlings. Commercially-available immunostrips (Agdia, Inc. or CSL Pocket Diagnostics) can be used for rapid detection of R. solanacearum in the field or lab. The screening tests are aimed at detection of R. solanacearum in plants, contaminated soil, and/or

4 water samples without identifying the race or biovar present. Some of the tests that are routinely applicable are described in the following sections.

2.3.1.1. Indicative symptoms and signs in infected plant materials

A plant sample showing wilting even under conditions of adequate soil moisture may be suspected to have R. solanacearum infection. However, it should be examined for soft rots of fleshy tissues and other symptoms inconsistent with R. solanacearum infection, in which case other causes may be suspected. It may also be possible that secondary infections by other organisms may overshadow the symptoms caused by R. solanacearum infection and should be taken into consideration. Cut stems or storage organs exhibiting bacterial slime upon pressing vascular bundles may be suspected to be infected by R. solanacearum or other bacteria but should be investigated further through isolation or other diagnostic procedures.

Observation of bacterial streaming from the cut end of plant tissue can provide a preliminary indication of bacterial wilt disease. Bacterial streaming can be observed using either one or both of the following steps:

i) cut a section of the stem of a freshly wilted plant, preferably near the base, and suspend it in a transparent glass, bottle or a beaker containing clean water. A cloudy white stream similar in appearance to the diffusion of a milk drop in water or smoke diffusing in air signifies the presence of R. solanacearum (Figure 1). Streaming is observed when the bacterial population in the vascular bundles, specifically the xylem, is high; low populations of the pathogen may not be visible to the naked eye.

Figure 1. Bacterial ooze from cut stem end looks like smoky strands to the naked eye.

ii) cut a thin section of the specimen to include vascular bundles, preferably near the base of the plant and place the section on a microscope slide with a few drops of water. Cover this slide with a coverslip and then view it under a microscope at low power (10X – 20X objective). For optimal visualization, use dark field illumination. If this feature is not available, an approximation can be fashioned by adjusting the source light to produce a dark background. This can be done by blocking the light so that only oblique rays reach the specimen, e.g. by placing a coin partially over the light source or under the stage. Streaming masses of bacteria signify the presence of R. solanacearum or another bacterium (Figure 2). Further investigation, such as culturing, immunoassay

5 and/or molecular analysis may be needed to identify the bacterial pathogen. It is important not to mistake the diffusion of particulate material such as latex, starch granules and plastids into the water for bacterial streaming. Unlike bacteria, these particulates vary in size and tend to exhibit random (Brownian) movement.

Figure 2. Bacterial ooze from plant tissue viewed under a microscope.

Note: (i) Particulate material such as latex, starch granules and plastids might be mistaken for bacteria. (ii) In some cases, bacterial masses may not be seen due to low numbers of cells, as is likely with cankers.

2.3.1.2. Isolating Ralstonia solanacearum from infected plant materials

A plant sample showing wilting even under conditions of adequate soil moisture can be subjected to isolation for R. solanacearum, whether there was bacterial streaming or not after undertaking the steps outlined above. Isolation would successfully distinguish this bacterium from others if carried out on diagnostic media. The procedure involves disinfection of the target plant material, preparation of a bacterial suspension from the target material and culturing on agar medium.

(i) Disinfection of infected plant material: After washing with water (preferably running from the tap), the selected plant material is immersed in bactericidal solutions, such as ethanol (70% or 95%) and sodium hypochlorite (0.5% NaOCl), for 2 to 3 minutes. This is to remove any saprophytic or epiphytic bacteria from plant surfaces.

(ii) Isolation: Initial isolation may be achieved through one of the following steps:

– Soaking surface-sterilized plant material in sterile distilled water to obtain a bacterial suspension – Macerating the surface-sterilized plant material in sterile distilled water to obtain a bacterial suspension

6 (iii) Culturing:

In all cases, care must be taken to utilize proper aseptic technique.

Initial culturing may be achieved through streaking a bacterial suspension (in water) onto a semi-selective medium, such as SMSA or tetrazolium chloride (TZC) agar plates (Appendix 1). In some cases, if the bacterial ooze is very profuse, culturing can be done by direct streaking of bacterial ooze and exudates on agar medium without going through the step of preparing a bacterial suspension in water.

The agar plates are then incubated at 28 - 30°C or at room temperature for 2-6 days. Plates should be incubated in an inverted position because water condensation may cause colonies to flow into each other, thereby limiting separation. Separately growing colonies can then be picked and sub-cultured onto fresh media to obtain pure cultures.

Appearance on SMSA. R. solanacearum appears as mucoid, whitish colonies after 48 hours. The colonies then develop blood red whorls in the center after further incubation (Figure 3). Colonies that are not R. solanacearum will be uniformly pigmented red.

Figure 3. Bacterial growth on streaked medium (left) and characteristic colonies of Ralstonia solanacearum on SMSA (right).

Appearance on modified tetrazolium chloride (TZC) medium. R. solanacearum colonies are characteristically mucoid and whitish, with a reddish-pink diffuse pigment (it does not diffuse into the medium). Often there is a brown discoloration of the medium around the colonies. Growth also occurs more quickly on TZC than on SMSA.

2.3.2. Advanced Screening in Specialized Laboratories

2.3.2.1. Classical analysis for biovar determination

Determination of the five biovars of R. solanacearum is done on the basis of carbon utilization in disaccharides and hexose alcohols (Hayward, 1964, Denny and Hayward, 2001). The disaccharides used are cellobiose, lactose and maltose, while the hexose alcohols are dulcitol, mannitol and sorbitol. Sterilized solutions (10% W/V) of these carbon sources are added to a basal medium (Appendix 1) before introduction of pure R. solanacearum

7 isolates. The hexose alcohols are relatively heat-stable and are therefore sterilised by autoclaving them at 121°C for 15 minutes. The disaccharides are heat labile and are therefore sterilised by filtration into pre-sterilised universal bottles or small flasks using 0.22 micron millipore membrane and syringe.

A 90 ml molten basal medium is cooled to approximately 50°C and then 10 ml of a given carbon source solution is added. After mixing, the medium is dispensed in 3-4 ml aliquots into previously sterilised test tubes (150 mm x 10 mm size), plugged with non-absorbent cotton. After setting, the media are stab-inoculated in duplicates with bacterial cultures of pure isolates. The cultures should be 24 to 48 hrs old on CPG (Appendix 1) and made into suspensions using sterile distilled water. A negative control is set up without any carbohydrate, where salicin, a non-carbohydrate, is most commonly used). A positive control is also included, where dextrose is most commonly used because it is utilized by all biovars. The tests are incubated at 28-30°C. This test can also be performed in a 96-well plate, as seen in Figure 4 (detailed instruction can be found in Appendix 2).

Reactions are observed and recorded over a period of several days, commonly up to 7 days. A colour change to yellow (acid pH < 6) indicates oxidation of the carbon source (Figure 4). This usually occurs within 3-5 days; those biovars capable of oxidizing disaccharides could take a few days longer to give a clear positive result. The inoculated tubes should also be compared with a non-inoculated control tube. Depending on the reaction produced, the biovar of a R. solanacearum isolate can be determined as shown in Table 2.

Figure 4. Carbon utilization test for biovars of Ralstonia solanacearum showing a positive result (yellow colour) and a negative result (green colour).

8 Table 2. Differentiation of Ralstonia solanacearum biovars based on utilization of various carbon sources Biovars Test 1 2* 3 4 5 Mannitol - - + + + Sorbitol - - + + - Dulcitol - - + + -

Lactose - + + - + Maltose - + + - + Cellobiose - + + - + Dextrose + + + + + *In order to differentiate between the sub-phenotypes 2A and 2T in biovar 2, D (+) trehalose, L (-) tryptophan and D-ribose are included in the panel of test carbon sources; strain 2A gives a negative result while strain 2T gives a positive result (as is the case with all the other biovars).

2.3.2.2. Detection of Ralstonia solanacerum in soil samples using a semi-selective medium, SMSA (as applied at KARI-NARL, Plant Pathology Section)

Soil samples are weighed into 10 g quantities. Each sample is suspended in 100ml of water or -1 phosphate buffer (4.26 g Na2HPO4 and 2.72g KH2PO4 l ; pH 7.2; PB) in a conical flask or a heavy gauge polythene bag. The sample is shaken vigorously for 2 minutes and the heavy soil particles were allowed to settle for about 2 minutes. Subsequently, the following steps are undertaken:

− Draw out a 1.5 ml aliquot from the suspension using a sterile pipette tip and a micro- pipette, being careful to avoid soil or debris. Transfer the suspension to a sterile Eppendorf tube; this forms the stock suspension (100). − Draw out a 100 µl aliquot from the stock suspension and put it in 900 µl of sterile distilled water in a sterile Eppendorf tube; mix thoroughly by pipetting in and out, while stirring simultaneously with the micro-pipette. This forms the first dilution of the stock suspension (10-1). − Dilute the 10-1 suspension by adding a 100 µl aliquot to 900 µl of sterile distilled water in a flat-bottomed tube to get a 10-2 suspension. − Continue the serial dilution procedure to get a 10-4 suspension (or higher dilutions if the samples are suspected to have high populations of R. solanacerum. − Mix the 10-4 suspension (or the highest dilution made) thoroughly using a pipette tip and draw out a 100 µl (0.1 ml) aliquot. − Place this suspension on a well-set agar plate containing SMSA and spread with a sterile bent glass rod to cover most of the surface of the medium (this is called ‘lawn-plating’). Be careful not to splash the suspension to the edge of the medium because counting the colonies at the edges can be difficult or impossible. Note that spreading should be done as soon as the suspension is placed on the medium to avoid ‘clumps’ of colonies. − Repeat the above two steps with the 10-2 and 100 suspensions. Other dilutions may be plated depending on whether the samples are suspected to have higher or lower populations of R. solanacerum. • Changing the pipette tip is not necessary between plating of any two dilutions of the same sample, PROVIDED THAT YOU START WITH THE LEAST

9 CONCENTRATED SUSPENSION per sample; however, a fresh sterile tip must be used for each separate sample.

Note: Whenever possible, it is advisable to prepare at least two plates for each dilution per sample, to cater for experimental error by using the mean of the counts in those plates.

Ø Positive control: Ensure that you dose a soil suspension with a confirmed isolate of R. solanacearum and carry out serial dilutions from 100 to 10-5. Plate the 10-1, 10-3 and 10-5 suspensions (one plate each is adequate). Also prepare another positive control of R. solanacearum in sterile distilled water and plate out the 10-1, 10-3 and 10-5 suspensions (one plate each is adequate).

− Incubate the plates at 30°C for 48-72 hrs. − Count the colonies that show typical R. solanacerum characteristics. The colonies should be fluidal and irregular in shape with a characteristic red or pinkish red centres and whitish periphery. These are best seen when observed against light. Opaque, dark pink/red colonies are likely to be seen but should be ignored during counting. − Calculate the population of R. solanacerum in the soil sample taking into consideration the dilution factor and the volumes used to make the stock suspension and the volume plated.

2.3.2.3. Detection of Ralstonia solanacearum in water samples using a semi-selective medium, SMSA (As applied at KARI-NARL, Plant Pathology Section)

The concentration of bacterial cells in water samples often must be enhanced to increase chances of detection. This can be done by centrifugation or, alternatively, through a settling- siphoning procedure. In each case, a water sample should first be passed through a fine sieve to remove large particles or particulate matter. All containers used must be clean.

Amplification of samples by centrifugation involves shaking a water sample thoroughly to homogeneity before drawing out 50ml as the working sample. This working sample is then centrifuged at 7000 x g at 4-10°C for 15 minutes. The supernatant is discarded and the resulting pellet re-suspended in 1ml of water.

In the absence of a centrifuge, the settling-siphoning procedure involves the following steps:

− After stirring the water to homogeneity, put 250 ml of the water sample in a clean sterile beaker (preferably a narrow beaker) and allow it to stand for about 30 minutes (or 15 minutes in a refrigerator set at 4-10°C); bacterial cells are expected to settle in the bottom part of the sample. − Decant the top half of the water without shaking or stirring. Then allow the water to stand for 15 minutes. − Siphon out the top part of the water sample with a sterile pipette (or micropipette with a sterile tip) to leave 25 ml. This forms the “concentrated suspension”. − Shake the sample briefly by hand and draw out a 1.0ml aliquot from the “concentrated suspension” using a sterile pipette tip and a micro-pipette, being careful to avoid too much soil or debris. Put this suspension in a sterile Eppendorf tube; this forms the “stock suspension”, which is easy to store and handle.

10 A 100 µl aliquot of the re-suspended pellet or 100 µl of the concentrated suspension is lawn- plated by placing it on a well-set agar plate containing SMSA and spread with a bent glass rod to cover most of the surface of the medium. It is advisable to prepare at least two plates for each concentrated suspension to cater for experimental error by using the mean of the counts in those plates. Care should be taken not to splash the suspension to the edge of the medium because counting of colonies at the edges can be difficult or impossible. Note that spreading should be done as soon as the suspension is placed on the medium to avoid ‘clumps’ of colonies.

Subsequently, − Incubate the plates at 30°C for 48 - 72 hours. − Count the colonies that show typical R. solanacerum characteristics: cream white colonies with pink or purplish pink centres. These are best seen when observed against light. Opaque, dark pink/red colonies are likely to be seen but should be ignored during counting. − Calculate the population of R. solanacerum in the water sample taking into consideration the volume of the “concentrated suspension,” the sample as originally drawn from the presented sample and the volume plated.

Ø Positive control: Dose a sterile distilled water sample with a confirmed isolate of R. solanacearum and carry out serial dilutions to 10-5. Plate the 10-1, 10-3 and 10-5 suspensions (one plate each).

Note: SMSA should be stored in an inverted position for 1-2 days before use to allow for surface drying; storage in the dark or diffuse light is recommended. Poor bacterial growth may result if the medium is stored for too long.

2.3.2.4. Molecular analysis for identification/detection of Ralstonia solanacearum

Plant, soil or water samples suspected to contain R. solanacearum can be subjected to molecular (DNA) analysis for identification or detection purposes. Application of molecular techniques to determine presence or absence of R. solanacearum, particularly when there are no clear symptoms or isolation is not guaranteed, may involve working with a suspected material directly to extract DNA. a) Extraction of Bacterial DNA for Molecular Analyses i) Preparation of Bacterial Cultures and Extraction of DNA

Culturing of Isolates: Pure isolates of R. solanacearum are cultured on Casamino Acids- Peptone-Glucose (CPG) agar: [(0.1%) peptone, (0.01%) casamino acids (Difco), (0.05%) glucose, and (1.5%) agar] and incubated at 28°C for 48 – 72 hours prior to the DNA extraction process.

DNA Extraction Method 1: Genomic DNA from bacterial cultures is extracted by heat lysis and selective precipitation of cell debris and polysaccharides with CTAB (cetyltrimethylammonium bromide), and precipitation of DNA with chloroform: octanol (a modification of the protocol of Wilson et al., 1987).

11 Briefly, sterile loops are used to remove 40 – 60 mg of bacterial biomass from each culture and deposited in a labeled 1.5 ml Eppendorf tube. Five hundred microlitres (500 µl) of warm (65°C ) CTAB (Appendix 3) extraction buffer are added into each tube and mixed gently. The tubes are incubated at 65°C for 1 hour with continuous gentle rocking (a Thermolyne 37600 mixer can be used).

The tubes are then cooled for 5 minutes at room temperature. Three hundred microlitres (300 µl) of chloroform:octanol (ratio 24:1, Appendix 3) are added to each tube and mixed by rocking gently for 10 minutes. The tubes are then centrifuged for 10 minutes at 3500 revolutions per minute (rpm); a Mini Spin Plus centrifuge may be used. The top aqueous layers are pipetted out into new labeled 1.5 ml Eppendorf tubes. Three hundred microlitres (300µl) of chloroform:octanol solution is added an additional time into each tube and the process is repeated.

Three microlitres (3 µl) RNase is added to each tube and incubated at 37°C for 1 hour. Three hundred microlitres (300 µl) of ice-cold absolute alcohol are added and the contents mixed gently. The tubes are centrifuged at 4000 rpm for 5 minutes. The resulting pellets are washed with 500 µl of Wash One (Appendix 3) for 20 minutes and then 500 µl of Wash Two (Appendix 3) for 10 minutes. The excess Wash Two buffer is drained from the tubes until dry. The pellets are then dissolved in 200 µl of 1X TE and refrigerated at 4°C until needed.

DNA Extraction Method 2: A loopful of a pure bacterial culture is suspended in 10 ml of phosphate buffer (PB) and homogenized before transferring 5 µl into a 200 µl PCR tube. This bacterial suspension is then covered with one drop of sterile mineral oil, boiled for 5 minutes, and kept in ice. ii) Extraction of Bacterial DNA in Plant Materials (Llop, 1999)

Suspect plant materials are first macerated in 50 mM phosphate buffer (4.26 g Na2HPO4 and -1 2.72 g KH2PO4 l ; pH 7.2) (PB) to homogeneity and then filtered before centrifuging at 10 000 g for 10 minutes. The resulting pellet is then re-suspended in 500 µl extraction buffer (200 mM Tris HCl pH 7.5, 250 mM NaCl, 25 mM EDTA, 0.5% SDS, 2% PVP), vortexed and left to stand for 1 hour at room temperature with continuous shaking. Centrifuging is then done at 5000 x g for 5 minutes before transferring 450µl of the supernatant into a new tube with 450µl isopropanol. The solution is mixed gently and incubated for 1 hour at room temperature. Subsequently, the mixture is centrifuged at 13000 x g for 10 minutes, the supernatant discarded and the pellet dried under a vacuum. Finally the pellet is resuspended in 100 µl water ready for further use. iii) Extraction of Bacterial DNA from Soil

Extraction Method 1 (Lee and Wang, 2000):

Soil samples, weighing 0.2 g, are suspended in 0.5 ml of distilled water by vortexing and allowed to stand for 5 minutes. The supernatant is then transferred to a new microfuge tube with an equal volume of phenol:chloroform:isoamyl alcohol (25:24:1, v/v/v; pH 7.6). These are then mixed by vortexing at the maximum speed for 1 minute and centrifuged at 16,000 x g for 10 minutes. After the aqueous phase is transferred to a new tube, 0.1 volume of 3 M sodium acetate (pH 5.2) and 0.6 volume of isopropanol are added. The solution is mixed well and centrifuged at 16,000 x g for 15 minutes. DNA pellets are washed with 70% ethanol

12 twice, vacuum dried, and resuspended in 20 µl of TE (10 mM Tris, 1 mM EDTA; pH 8.0) buffer and stored at -20ºC until needed.

Extraction Method 2 (Pradhanang et al., 2000): Soil samples, weighing 10 g, are suspended in 100 ml PB and shaken vigorously for 2 minutes. The heavier soil particles are allowed to settle for 2 minutes. Then, 90 ml of the supernatant is removed, mixed with 10 ml of 0.5M NaOH and boiled for 4 minutes. This becomes a DNA suspension that is ready for further use or can be stored at -20ºC until needed.

b) Polymerase Chain Reaction-Based Identification/Detection of Ralstonia solanacearum

DNA Analysis Method 1 [using primer system CHR5 (Grover et al., 2011)]: PCR is performed using R. solanacearum-specific primers CHR 5 (forward [5-TCG TGT GTC GAA AGA GTG CT-3] and reverse [5-CTT GTC TGC CTC GAG TTG TG-3] in 25 µl reaction mixtures containing 1X PCR buffer (Applied Biosystems, USA), 2.0 mM MgCl2 concentration, 200 mM deoxynucleoside triphosphate (dNTP), 30 pM primer concentration, 1.0 U Taq DNA polymerase (Applied Biosystems, USA), and 50 ng DNA (or 5 µl of DNA extraction suspension). The PCR regime is as follows: 2 minutes denaturation at 95ºC followed by 40 cycles of 5 seconds denaturation at 95ºC, 30 seconds annealing at 60ºC and 30 seconds elongation at 72ºC and one cycle at 72ºC for 10 minutes. A Perkin Elmer thermal cycler (GeneAmp PCR System 9700) may be used. When the resulting PCR products are analyzed on 1% agarose gels in TAE buffer (10 V/cm may be used), a single band of approximately 730 bp should be expected after staining in ethidium bromide solution (1 µg ml−1), confirming the presence of R. solanacearum.

DNA Analysis Method 2 [using primer system AU759/AU760 (Lin et al., 2009)]: PCR is performed on the sample using R. solanacearum-specific primer pair AU759f (5′-GTC GCC GTC AAC TCA CTT TCC-3′) and AU760r (5′-GTC GCC GTC AGC AAT GCG GAA TCG-3′) as described by Opina et al. (1997). A 25µl reaction mixture contained 1x PCR buffer [10 mM Tris–HCl (pH 9.0), 50 mM KCl, and 0.1% Triton X-100], 1.5mM MgCl2 concentration, 0.05mM of each dNTP, 1 pM of each primer, 2 U of Taq DNA polymerase (Promega, Madison, USA), and 5µl of boiled bacterial suspension (or 5µl of DNA extraction suspension). The following PCR amplification regime is used: denaturation at 94°C for 3 minutes, annealing at 53°C for 1 minute, and extension at 72°C for 1.5 minutes, followed by 30 cycles of 94°C for 18 seconds, 60°C for 18 seconds and 72°C for 18 seconds and a final extension of 72°C for 5 minutes. A specific PCR product of 282 bp is expected. This can be visualised under UV light after electrophoresis in 1.5% agarose and staining in ethidium bromide solution (1 µg ml−1). 2.4. Confirmation

Taking all the sampling, despatch and receipt circumstances, and the subsequent isolation and analysis, the results obtained should be scrutinized by a practising specialist (plant pathologist/bacteriologist) with experience in interpretation of results involving analysis for Ralstonia solanacearum. In arriving at a conclusion, the specialist should consider at least one confirmatory test carried out on the sample. Where ambiguity abounds, more tests must be considered. Thereafter, a comprehensive report authenticized by the head of the laboratory should be prepared.

13 Contact Persons and Laboratories

Dr. Zachary M. Kinyua Prof. Sally Miller Plant Pathology Section, Department of Plant Pathology National Agricultural Research Laboratories, The Ohio State University Kenya Agricultural Research Institute Ohio Agricultural Research and PO Box 14733-00800, Nairobi, KENYA Development Center 1680 Madison Ave. Email : [email protected] Wooster, OH USA 44691 Tel: +254-(0)20-4444142/4443957 cell: +254-(0)733-999444 Email: [email protected] Fax: +254-(0)20-4443926 Phone: +1 330-263-3678 Fax: +1 330-263-3841 Cell: +1 330-466-5249

2.5. Communication

Results obtained from the analyses, samples obtained and any relevant policy formulation or implementation from an agency should be summarized into a report that can easily be understood by the client and/or technical advisors of the client. Positive overall results should be accompanied by advice on what needs to be done to reduce the existing effect and potential risk of the disease. Negative results should be communicated with advice on what should be done to prevent possible future infestations.

2.6. Sample Destruction

All materials (plant, soil or water) and/or cultures resulting from isolation and/or supplies used in the analyses involving Ralstonia solanacearum should be destroyed by autoclaving at a minimum of 121°C (15 psi) for at least 15 minutes before being disposed. All apparatus, equipment and tools should be sterilized appropriately soon after use to prevent any future contamination.

14 Appendix 1: Media for isolation and cultivation of Ralstonia solanacearum

Modified SMSA for isolation of Ralstonia solanacearum (As used at KARI-NARL, Plant Pathology Section)

Preparation of the basal medium

For 1000ml For 500ml For 250ml Casamino acids (Difco) 1.0g 0.5g 0.25g Bacto-Peptone (Difco) 10.0g 5.0g 2.50g Glycerol 5.0ml 2.5ml 1.25ml Bacto-Agar (Difco) 15.0g 7.5g 3.75g Distilled water 1000ml 500ml 250ml If Bacto-Agar (Difco) is not available, use Oxoid Agar No. 1 – but the growth of R. solanacearum will be slower, typical colonies may take 1-2 days longer to express themselves, and the colony coloration may be lighter and more diffuse. It is advisable to dispense the basal medium into 250ml (preferable) or 500ml quantities before autoclaving. • Autoclave the above ingredients at 121ºC for 15 minutes • Cool to about 40-45ºC before adding the additives as shown below. • Pour the homogenized medium into sterile Petri dishes.

Preparation of additive solutions (filter-sterilized) and incorporation into basal medium

Stock solution concentration Required Amount to add to basal Additive concentration medium in basal medium 250 ml 500ml Crystal violet 1% (0.1g in 10mls water) 5mg/litre 125µl 250µl (Sigma) Chloramphenicol 1% (0.1g in 10mls Methanol) 5mg/litre 125µl 250µl (Sigma C-3175) Penicillin G 0.1% (0.01g in 10mls water) 0.5mg/litre 125µl 250µl (Sigma P-3032) Bacitracin 1% (0.1g in 10mls Methanol) 25mg/litre 625µl 1250µl (Sigma B-0125) Tetrazolium salts 1% (0.1g in 10mls water) 50mg/litre 1250µl 2500µl (Sigma) Polymixin B 1% (0.1g in 10mls water) 100mg/litre 2500µl 5000µl Sulphate (Sigma P-1004)

15 CPG Medium for cultivation of Ralstonia solanacearum

Casamino acids (Difco) 1.0g Bacto-Peptone (Difco) 10.0g Glucose 10.0g Bacto-Agar (Difco) 18.0g Distilled water 1000ml

• Autoclave the above ingredients at 121ºC for 15 minutes and cool to about 40-45ºC before pouring. • Pour the homogenized medium into sterile Petri dishes. ======

Kelman’s Tetrazolium Medium

Casamino acids (Difco) 1.0g Bacto-Peptone (Difco) 10.0g Dextrose 5.0g Bacto-Agar (Difco) 15.0g Distilled water 1000ml

• Dissolve the ingredients and sterilize by autoclaving at 121ºC for 15 minutes. • Cool to about 45-50ºC and add filter-sterilized tetrazolium chloride (Sigma) to obtain a final concentration of 50mg per litre (i.e. 5mls of a 1% solution to each litre of medium). • Pour the homogenized medium into sterile Petri dishes.

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Carbohydrate Medium for Biovar Determination (After Hayward, 1964; Hayward, 1976)

Basal medium preparation NH4H2PO4 1g KCl 0.2g MgSO4.7H2O 0.2g Peptone 1.0g Bromothymol blue 10-20ml Distilled water 1L

Adjust pH to 7.0 with 40% NaOH. Then add 3.0g of Agar. Dispense in 45ml aliquots in 50ml flasks plugged with cotton wool. Sterilize by autoclaving at 121ºC for 15 minutes.

16 Appendix 2: Ralstonia biovar testing

Basal media preparation: 1000 ml 700 ml* Ammonium dihydrogen phosphate (NH4H2PO4): 1.0 g 0.70 g Potassium chloride (KCl): 0.2 g 0.14 g Magnesium sulphate (MgSO4. 7H2O): 0.2 g 0.14 g Peptone: 1.0 g 0.70 g Bromothymol blue: 0.03 g 0.021 g Agar: 3.0 g 2.10 g Water: 1.0 L 700 ml

* It is advised to prepare the volume of medium that can easily be divided into seven equal parts, such as: 700 ml or 350 ml.

Bring the medium to boil with constant stirring. Raise the pH of the medium to 7.0 – 7.1 by drop wise addition of 1.0 N sodium or potassium hydroxide. The medium turns green. Divide the medium into seven containers, each container with 90 ml of medium. Autoclave at 121°C, 15 psi for 20 minutes.

Sugar/alcohol solution preparation: Dissolve of 1g of cellobiose, lactose, maltose, dulcitol, mannitol and sorbitol in 10 ml distilled water separately. Filter-sterilize them using 0.22µm filters. Alternatively, these solutions can be sterilized by boiling them in water bath for 20 minutes for three successive days.

Preparation of media: After autoclaving, cool the basal medium to about 65 0C and mix the 10 ml prepared sugar or alcohol solutions each with 90 ml basal medium. For the control, add 10 ml sterile water (without sugar/alcohol) to 90 ml basal medium.

Allocation of media: The media now can be dispensed either on 96 well plates or 15 ml tubes. 96-well plate: take a sterile 96-well plate (or sterilize with UV-light under a hood) and fill each row of the plate with different sugar/alcohol-amended medium. Pour 150 µl of medium in each well. Seal the plate with sterile (UV-sterilized) plastic tape. For convenience, you can fill the plate as shown in the figure below:

1 2 3 4 5 6 7 8 9 10 11 12 A Cellobiose B Lactose C Maltose D Control E Dulcitol F Mannitol G Sorbitol H Empty

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To speed up the process, make a boat of aluminum foil and sterilize it by spraying alcohol followed by flaming. Now pour the medium into the boat and use a multichannel pipet to dispense it in all 12 wells at once. Use sterile tips. Clean the boat thoroughly and sterilize it again before using for other media.

15ml tube: dispense 3 ml of sugar/alcohol-amended medium in each tube and close the tube tightly with cotton plug or screw cap.

Inoculum preparation: Grow isolates in CPG or TTC medium for 48 hrs at 28 0C. Take a loop-full of bacteria and mix it to 1ml sterile water in a 1.5 or 2.0 ml centrifuge tube (Eppendorf). The suspension should be cloudy. Aseptically dispense on e small drop (~7 µl) of the bacterial suspension in each well of a column.

For 15ml tubes, 20 µl of bacterial suspension should be used. Incubate the plate/tubes at 28 0C.

Observation: Results can be observed in 4-7 days in 96-well plates and 3-4 weeks in 15 ml tubes. If an isolate utilizes a sugar or alcohol, the color of the medium changes to yellow, otherwise it remains blue-green. Biovar determination: BV BV BV BV BV 1 2 3 4 5 A B C D E F G H

18 Appendix 3: Reagents for DNA analysis

1. CTAB extraction buffer1 Stock Final concentration Reaction volume (10ml) 50ml Double distilled water - 6.5ml 32.5 1M Tris – 7.5 100mM 1.0ml 5ml 5M Nacl 700mM 1.4ml 7ml 0.5M EDTA – 8.0 50ml 1.0ml 5ml CTAB2 1% 0.1g 0.5g 14M BME3 140mM 0.1ml 0.5ml 1 Use freshly made; warm buffer to 60 – 65oC before adding the CTAB and BME. 2 CTAB – mixed alkyltrimethyl – ammonium bromides (Sigma m – 7635) 3 Add BME (B-mercaptoethanol) just prior to use, under a fume hood.)

2. Chloroform/Octanol mixture (24:1) Stock (100ml): 96ml Chloroform + 4ml Octanol

3. Wash 1. (76% Ethyl alcohol, 0.2 M NaOAC) Stock (100ml) Absolute ethanol 76ml 2M NaOAC 8ml Distilled water 16ml Dissolve sodium acetate in double distilled water. Sterilize by autoclaving.

4. Wash II (76% Ethyl alcohol, 10mM NH4OAC) Stock (100ml) Absolute ethanol 76ml 1M NH4OAC 1ml Distilled water 23ml

5. Tris HCl/EDTA buffer (TE buffer) Stock (100ml) 1M Tris.HCl –pH 7.4 1ml 0.5 M EDTA 0.2ml Sterile distilled water 98.8ml

6. TAE buffer (Tris HCl, glacial acetic acid, EDTA) Stock (50X) Trizma base 242g EDTA (0.5M) 57.1ml Glacial acetic acid 100ml Mix Tris with double distilled water by stirring, add EDTA and acetic acid then bring the final volume to 1 litre with double distilled water.

19 Appendix 4: Bibliography

Ajanga, S. 1993. Status of bacterial wilt in Kenya. In: Bacterial wilt. Hartman, G. L. and Hayward, A. C. (eds.), pp 338-340. ACIAR proceedings No.45, Canberra, Australia.

Alacho, F.O. and Akimanzi, D.R. 1993. Progress, achievements and constraints on bacterial wilt control in Uganda. pp 32-41 In: Workshop on Bacterial wilt of potato caused by Pseudomonas solanacearum, Bujumbura, Burundi, Feb.22-26, 1993.

Ateka, E.M., Mwang’ombe, A.W. and Kimenju, J.W. 2001. Reaction of potato cultivars to Ralstonia solanacearum in Kenya. African Crop Science Journal 9: 251-256.

Barton, D., Smith, J.J. and Kinyua, Z.M. 1997. Socio-economic inputs to biological control of bacterial wilt disease of potato in Kenya. ODA RNRRS crop protection project R6629. NR International, United Kingdom. 23pp.

Coelho N. and Nutter, F. 2005. Bacterial wilt in Amazon State. In: Bacterial Wilt Disease and the Ralstonia solanacearum Species Complex. Allen, C., Priou, P. and Hayward, A.C. (eds.). APS Press, St. Paul, Minnesota USA.

Denny, T.P. and A.C. Hayward. 2001. Gram Negative Bacteria. In Laboratory Guide for Identification of Plant Pathogenic Bacteria, edited by N. W. Schaad, J. B. Jones and W. Chun. St. Paul, Minnesota: APS Press.

Elphinstone J.G., Hennessy J., Wilson J.K., Stead D.E. 1996. Sensitivity of different methods for the detection methods for the detection of Ralstonia solanacearum in potato tuber extracts. Bulletin OEPP/EPPO Bulletin 26: 663-78.

Engelbrecht M.C. 1994. Modification of a semi-selective medium for the isolation and quantification of Pseudomonas solanacearum. ACIAR Bacterial Wilt Newsletter 10: 3-5.

Fegan, M. and Prior, P. 2005. How complex is the "Ralstonia solanacearum species complex"? Pages 449-461 in: Bacterial Wilt Disease and the Ralstonia solanacearum Species Complex. C. Allen, P. Prior, and A. C. Hayward, eds. American Phytopathological Society, St. Paul, MN.

Grover, A., A. Grover, S.K. Chakrabarti, W. Azmi, D. Sundar and S.M.P. Khurana. 2011. Identification of Ralstonia solanacearum using conserved genomic regions. Int. J. Biotechnol. Mol. Biol. Res. 2: 23-30.

Hayward, A. C. 1991. Biology and epidemiology of bacterial wilt caused by Pseudomonas solanacearum. Annu. Rev. Phytopathol. 29:65-87.

Kelman, A. 1954. The relationship of pathogenicity of Pseudomonas solanacearum to colony appearance in a tetrazolium medium. Phytopathology 44:693-695.

Lee, Y-A and C.-C. Wang. 2000. The design of specific primers for the detection of Ralstonia solanacearum in soil samples by polymerase chain reaction. Bot. Bull. Acad. Sin. 41:121- 128.

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Lin, C.-H., Hsu, S.-T., Tzeng, K.-C., and Wang, J.-F. 2009. Detection of race 1 strains of Ralstonia solanacearum in field samples in Taiwan using a BIO-PCR method. European Journal of Plant Pathology 124:75-85.

Llop, P., P. Caruso, J. Cubero, C. Morente, M.M. López. 1999. A simple extraction procedure for efficient routine detection of pathogenic bacteria in plant material by polymerase chain reaction. Journal of Microbiological Methods 37: 23–31.

Pradhanang, P.M., J.G. Elphinstone and R.T.V. Fox. 2000. Sensitive detection of Ralstonia solanacearum in soil: a comparison of different detection techniques Plant Pathology 49: 414-422.

Prior, P. and Fegan, M. 2005. Recent developments in the phylogeny and classification of Ralstonia solanacearum. Acta Hort. 695:127-136.

Schaad, N. W., Jones, J. B., and Chun, W. 2001. Laboratory Guide for the Identification of Plant Pathogenic Bacteria, 3rd Ed, APS Press, St. Paul, MN.

Schönfeld, J., H. Heuer, J. D. van Elsas and K. Smalla. 2003. Specific and sensitive detection of Ralstonia solanacearum in soil on the basis of PCR amplification of fliC fragments. Appl. Environ. Microbiol. 69: 7248. DOI:10.1128/AEM.69.12.7248-7256.2003. van Elsas, J. D., Kastelein, P., de Vries, P. M., and van Overbeek, L. S. 2001. Effects of ecological factors on the survival and physiology of Ralstonia solanacearum bv. 2 in irrigation water. Can. J. Microbiol. 47:842-854.

Wilson, K. 1987. Preparation of genomic DNA from bacteria, p. 210–212. In F. M. Ausubel, R. Bent, R. E. Kingston, D. D. Moore, J. A. Smith, J. G. Seidman, and K. Struhl (ed.), Current protocols in molecular biology. Greene and Wiley, New York, N.Y.

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