Detection of Plant-Pathogenic Bacteria in Seed and Other Planting Material, Second Edition

CHAPTER 5

Detection of Xanthomonas translucens in Wheat Seeds Etienne Duveiller1 and Claude Bragard2 1. CIMMYT, Global Wheat Program, El Batán, Apdo Postal 6-641, 06600, Mexico D.F., Mexico.

2. Université catholique de Louvain, Earth & Life Institute, Croix du Sud, 2bte3, B-1348 Louvain-la-Neuve, Belgium. and specificity make all these techniques difficult to apply for routine detection of X. translucens pv. undulosa in seed health laboratories. In the CIMMYT seed health laboratory in Mexico, the immunofluorescence (IMF) serological method developed by Duveiller and Bragard (9) is currently the ISO/IEC (International Organization for Standardization, Switzerland) 17025 accredited protocol for this pathogen (24). Real-time PCR assays for rapid and reliable detection of the pathogen in wheat seed are not available yet, partly because only a few studies have been conducted on the pathogen in recent years. New efforts in multilocus sequence analysis (MLSA) and in sequencing closely related X. translucens pathovars infecting small grains should lead, in the near future, to the development of reliable and rapid PCR-based seed health assays for X. translucens.

I. Introduction Black chaff or bacterial leaf streak, caused by Xanthomonas translucens (ex Jones et al., 1917, Vauterin et al., 1995) (17, 32), is the most common wheat bacterial disease. Black chaff has been reported worldwide, especially from warm and humid locations. It is usually considered unimportant, but it is seedborne and thus a constraint for international germplasm exchange (7, 13). Several countries include X. translucens on their quarantine list, which requires effective protocols for detection of the bacterium in seed.

Yield losses as high as 20–40% have been reported (11,13). Controlling black chaff is not easy and relies essentially on using pathogen-free seed and resistant varieties (12, 22, 30). Whereas a few X. translucens pathovars can infect cereals (5, 10), others are more specific to grasses or are even able to infect woody plants (16, 26).

The barley pathogen, X. translucens pv. translucens, is restricted to barley and does not infect wheat, whereas the wheat pathogen, X. translucens pv. undulosa (Xtu) (31), infects both wheat and barley. Strains of X. translucens pv. cerealis (from Bromus spp.) and X. translucens pv. secalis (from rye) can be pathogenic to wheat (4, 5, 13, 26). Several techniques have been developed to assay wheat seed for infection by bacteria, including grow-out tests and inoculation of plants (23), agar plating on semiselective media (6, 19, 28), biochemical tests and fatty acid profiles (4, 13), serological methods (2, 3, 9), and PCR assays based on rDNA spacer sequences (21). However, cost, time, space, throughput,

II. Symptoms X. translucens pv. undulosa can induce symptoms on leaves and spikes of wheat and triticale (Figs. 5.1 and 5.2). Initially, lesions are water-soaked and produce a honeylike exudate under humid conditions (Fig. 5.1A). If undisturbed, the exudate hardens into yellowish, resinous granules, which stud the surfaces of lesions and are easily detached (Figs. 5.1D and 5.2A). Frequently, these drops coalesce when there is dew, rain, or guttation water to form conspicuous milky drops that may later spread over the leaf surface and dry down to thin, grayish, almost transparent flakes (Fig.

Fig. 5.1. Symptoms caused by Xanthomonas translucens pv. undulosa on wheat and triticale leaves. A, Fresh, milky exudate and water-soaking observed on triticale leaves.

B, Spreading exudate producing flakes on leaves with bacterial leaf streak. C, Translucent lesion in the middle of a durum wheat leaf at the location where dew is present on the leaf in the mornings. D, Typical bacterial streak of wheat with translucent stripes and exudate. E, Inconspicuous lesions on wheat. (Courtesy E. Duveiller—© APS) 27

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Part II / Detection of Plant-Pathogenic Bacteria in Cereals, Grain, Legumes, Grasses, and Forage

5.1A and 5.1B). Humidity favors the production of exudate, and thus lesions may appear more clearly in the middle of the leaf where bending allows dew to persist in the mornings (Fig. 5.1C).

Typical symptoms on the leaf include elongated, light-brown lesions, each several centimeters long, which are initially distinct (Fig. 5.1D) but later coalesce to cover larger areas of the leaf (Fig. 5.1E).

The culm, leaves, rachis, glumes, and awns can become infected, and symptoms on wheat have been reported to vary with the environment, cultivar, and disease severity. In farmers’ fields, symptoms are generally not detected at the seedling stage. X. translucens pv. undulosa can promote frost damage on young leaves in the spring (1) and survives epiphytically on symptomless leaves (7, 8). On spikes (Fig. 5.2), typical black chaff symptoms include discoloration of the peduncles and alternating bands of healthy and diseased tissues on the awns. These symptoms should not be confused with melanic reactions on ears and nodes known as pseudo black chaff, induced in genotypes carrying the gene Sr2 (13).

III. Epidemiology Seed is the most important source of primary inoculum for X. translucens pv. undulosa in wheat, and large-scale transmission of black chaff is due to the seedborne nature of the pathogen (Fig. 5.3). Depending on storage conditions, the bacterium, which is mostly located in the external seed coats, may survive in seed for more than 5 years. Recovery rate of the bacterium from seed may be reduced by 75% after 6 months, and by 99% after 3 years (13).

X. translucens pv. undulosa has a low seed transmission rate; i.e.,

low levels of seed contamination (<1,000 CFU/g seed) will normally not result in disease in the field (29). The bacterium survives poorly in soil and in decomposing plant stubble but can survive on grasses and weeds (13). Bacterial leaf streak outbreaks are characterized by sporadic epidemics and greater incidences in breeders’ plots. Moisture facilitates pathogen release from infected plant tissues and dispersal. Although X. translucens pv. undulosa tolerates a wide range of temperatures (15–30°C), temperature has a direct effect on disease severity. Pathogen multiplication in the leaf tissue is not limited by dry air conditions. Thus, it is not surprising to observe black chaff in western Siberia or Kazakhstan, where rainfall is rare during the summer season and frost often occurs. Black chaff is endemic in parts of Argentina, Brazil, and Uruguay (13), and has reemerged as a concern in the Great Plains region of the United States in recent years (18), whereas the bacterium has become rare in Mexico in the last decade based on our own observations.

IV. Disease Management Several methods have been described to control and limit the spread of X. translucens pv. undulosa, including dry heat seed treatment, seed treatment with acidified cupric acetate (14), cupric hydroxide, formalin (0.8%), or Guazatine Plus (3 ml/kg seed) (13).

Although the effects of these treatments are significant in controlling black chaff in the field, they are generally not used by farmers because of the costs. Using resistant varieties reduces the risk of disease outbreaks and severity. Thus, there is great need to identify new resistance sources (12, 17, 24, 25). However, because varietal resistance to black chaff is incomplete (12, 13, 18), control strategies require seed production in disease-free areas and the use of seedlots that have tested negative for X. translucens pv. undulosa.

V. Protocols for the Detection of X. translucens pv. undulosa in Wheat Seed A. Dilution Plating on Semiselective Agar Medium and Pathogenicity Test

Fig. 5.2. Symptoms caused by Xanthomonas translucens pv. undulosa on wheat spikes. A, Peduncle with resinous granules resulting from severe bacterial leaf streak infection. B, Typical black chaff symptoms with discoloration of the peduncle and alternating bands of healthy and diseased tissue on the awns.

(Courtesy E. Duveiller—© APS)

A seed wash test on semiselective agar media after 10-fold serial dilution plating (Fig. 5.4A) is the most commonly used nondestructive procedure in seed certification programs (14). The number of CFU per gram of seed gives an estimate of the number of living bacterial cells present in the sample. The best estimate is obtained by counting colonies on plates where the number of colonies ranges between 50 and 300 (Fig. 5.4B). Hence, when x grams of seed are washed in x ml (wt/vol = 1:1) of cold saline solution (0.85% NaCl containing 0.02% vol/vol Tween 20), and 0.1 ml is plated onto agar media: CFU/gram = nd x 10(d+1) where n represents the number of colonies counted on the medium at dilution, d.

The method used for the detection of X. translucens pv. undulosa from wheat seeds is as follows: 1. Add 120 ml of cold sterile aqueous saline to 120 g seed (approximately 3,000 seeds).

2. Shake vigorously for 3–5 min, let settle for 1 min, and dilute the suspension serially to 10-3.

3. Transfer 0.1 ml onto each of three agar plates of WBC (Appendix) semiselective medium per dilution, and spread the aliquot with a sterilized L-shaped rod.

4. Count colonies on the agar medium after plates have been incubated for 4 days at 30°C. 5. Calculate bacteria (CFU) per gram of seed.

WBC (Appendix) is the preferred medium agar used at CIMMYT, Mexico (5). WBC contains cycloheximide to reduce

Chapter 5 / Detection of Xanthomonas translucens in Wheat Seeds

fungal growth, boric acid (0.75 g/liter), and cephalexin (10 mg/liter), but no gentamycin. Other semiselective media include the lactose-based XTS agar (28) and KM-1 agar (19) (Appendix).

Representative colonies can be subcultured and tested for pathogenicity on wheat. Healthy, susceptible wheat plants (4–5 weeks old) produced in a greenhouse are inoculated. Usually the wheat

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stem or a leaf is pricked with a needle that has been passed through a pure colony (24–36 h culture) of the putative X. translucens pv. undulosa culture. Plant stems or young leaves can also be infiltrated with a cell suspension (~1 × 106–107 CFU/ml). Plants are then incubated for 48 h by covering them with a plastic bag at 22°C (Fig. 5.5A). Symptoms become visible 4–5 days after inoculation (Fig. 5.5B).

Fig. 5.3. Life cycle of Xanthomonas translucens pv. undulosa and possible ways the pathogen may spread. (Courtesy E. Duveiller—© APS)

Fig. 5.4. A, Xanthomonas translucens pv. undulosa detection using wheat seed washing and a dilution plating technique. B, Growth of X. translucens pv. undulosa on two replicate Petri dishes of WBC agar medium after a wash of infected wheat seed was serially diluted and plated on the agar medium. (Courtesy E. Duveiller—© APS)

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Part II / Detection of Plant-Pathogenic Bacteria in Cereals, Grain, Legumes, Grasses, and Forage

B. Immunofluorescence (IF) 1. Pipette 40 µl of seed wash water directly from the flask into a 6-mm well on a multiwindow slide (Biomérieux Marcyl’Étoile, France), and then fix the bacteria on the slide using hot air from a hair dryer (Fig. 5.6A).

2. Expose each well for 60 min to monoclonal antibody AB3B6 (Université catholique de Louvain, Belgium) diluted 100 times in phosphate-buffered saline (PBS; 8 g of NaCl, 2.7 g of Na2HPO4·2H2O, and 1 liter of distilled water, pH 7.2; use 20 µl of antibody per well).

3. Rinse the wells with PBS and expose them for 30–60 min in the dark to a mouse anti-rat MAb conjugated with fluorescent isothiocyanate MARM4-FITC (IMEX, UCL, Brussels) diluted 1:100 in PBS. The optimum dilution will vary with each batch of conjugated antibody and, therefore, must be determined empirically using a known positive sample. Use 20 µl of antibody per well.

4. Rinse the wells with PBS. 5. Add three drops of buffered glycerin (100 mg of diphenylamine in 10 ml of PBS, pH 9.6, and 90 ml of glycerol); apply a cover glass.

6. Observe under immersion oil using a microscope equipped with a high-pressure mercury ultraviolet lamp HBO-50 and Carl Zeiss filter combination ×1,000) (Fig. 5.6B). If the preparation cannot be observed on the same day, the multiwindow slides can be stored in the dark for later observation.

C. PCR Assay–Based Protocols PCR assay–based protocols are not available yet for routine wheat seed health testing X. translucens pv. undulosa but should be developed in the near future. PCR primers designed to exploit the variability associated with two short DNA sequences situated in the spacer segment between the 16S and 23S rRNA genes, and flanking an alanine-tRNA gene, have been proposed (21). They amplify a 139-bp fragment from strains of leaf streak pathogens from cereals, X. campestris pvs. cerealis, secalis, translucens, and undulosa, but also X. campestris pathovars pathogenic on forage grasses. Amplification of extracts of two seedlots known to be contaminated with a translucent leaf streak pathogen produced the 139-bp amplicon; 4 of 27 additional seedlots with unknown levels of leaf streak pathogens were positive for this amplified fragment

(21). The assays proved to be fast and relatively sensitive (2 × 103 CFU/g seed), indicating the technique might be useful for detecting pathogens in cereal seedlots. However, more specific primers are needed to avoid false positive reactions. Likewise, further studies are needed to unravel the status of X. translucens found on pistachio or asparagus (15, 26).

APPENDIX 1. KM-1 (19) Lactose D(+) trehalose Thiobarbituric acid K2HPO4 KH2PO4·7H2O Yeast extract NH4Cl Bacto agar (Difco) Distilled water

10.0 g 4.0 g 0.2 g 0.8 g 0.8 g 1.0 g 1.0 g 15.0 g 1.0 liter

Before adding the agar, dissolve the ingredients completely and adjust pH to 6.6 using 1 N NaOH. After autoclaving, cool the medium to 50°C. Then add: Cycloheximide (dissolved in 95% ethanol) 100.0 mg Ampicillin (dissolved in 50% ethanol) 1.0 mg Tobramycin (dissolved in 50% ethanol) 8.0 mg

2. Wilbrink’s Boric Acid-Cephalexin (WBC) Medium (6, 27) Bacto peptone Sucrose K2HPO4 MgSO4·7H2O Na2SO3 (anhydrous) Agar Distilled water

5.00 g 10.00 g 0.50 g 0.25 g 0.05 g 15.00 g 850.00 ml

Mix with the following solution (autoclaved separately): Boric acid 0.75 g Distilled water 150.00 ml

After cooling to 45°C, add: Cycloheximide (in 2 ml of 75% ethanol) 75.00 mg Cephalexin (1 ml of a 10 mg/ml stock 10.00 mg solution in 75% ethanol).

Fig. 5.5. A, Pots with Xanthomonas translucens pv. undulosa–inoculated wheat plants covered with plastic bags to produce humid chambers. B, Induced leaf streak symptoms with typical water-soaking. (Courtesy E. Duveiller—© APS)

Chapter 5 / Detection of Xanthomonas translucens in Wheat Seeds

3. XTS (28) Glucose Nutrient agar (Difco)

LITERATURE CITED 5.0 g 23.0 g

After autoclaving and cooling to 45°C, add: Cycloheximide (20 ml of a 100 mg/ml 200.0 mg stock solution in 75% ethanol) Cephalexin (1 ml of a 10 mg/ml 10.0 mg stock solution in 75% ethanol) Gentamycin (0.8 ml of a 10 mg/ml 8.0 mg stock solution in 75% ethanol)

4. Buffer: Phosphate-Buffered Saline (PBS) (0.01 mol/liter, pH 7.2) (20) Na2HPO4·12H2O Na2HPO4·2H2O NaCl Distilled water

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1.15 g 0.40 g 8.00 g 1.00 liter

Fig. 5.6. A, Immunofluorescence microscope. Seed wash samples are fixed onto a multiwindow slide with a hair dryer (not shown). B, Epifluorescence microscope view for the observation of immunofluorescence; fluorescent rods of Xanthomonas translucens pv. undulosa are observed under immersion oil. (Courtesy E. Duveiller—© APS)

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