Xanthomonas axonopodis pv. phaseoli
(bean blight)

Incidence

X. axonopodis pv. phaseoli (Xap) and X. axonopodis pv. phaseoli var fuscans (Xapf) is extensively seedborne on Phaseolus spp. A survey of navy bean (P. vulgaris) seeds showed that approximately 35% had internal contamination with X. axonopodis pv. phaseoli (Saettler and Perry, 1972).  34 bean seed lots were collected in Paraná, Brazil from 1998 to 1999 and, when studied, 50% of the seedlots were infected with Xap with incidences ranging from 0.1 to 1.7% (Torres et al. 2009). Assays conducted on seed collected from naturally contaminated commercial fields in Serbia indicated presence of Xap in 20 of the 23 cultivars from which collections were made from (Popovic et al., 2010). Five out of seven seed samples collected in Egypt tested positive for the presence of Xap (Abd-Alla et al., 2010). Incidences of seed infection by X. axonopodis pv. phaseoli as high as 16.1%, with populations of the bacterium averaging between 100,000 and 1,000,000,000 c.f.u./100 seeds, were recorded in farmers commercial and research seeds in Africa (Opio et al., 1993). Approximately 1 in 10,000 seeds is capable of causing an outbreak of blight (Sutton and Wallen, 1970), or 1000 to 10,000 bacteria per seed is the minimum needed to produce infected plants under field conditions (Weller and Saettler, 1980).
Infection of flower buds and young pods can result in the transmission of X. axonopodis pv. phaseoli through the vascular system to the seed, leading to internal infection (Aggour et al., 1989). X. axonopodis pv. phaseoli is a systemic pathogen that penetrates the ovule through the funiculus. It enters the sutures of the pod from the vascular system of the pedicel and passes into the raphe leading into the seed coat. Bacteria also may enter though the micropyle (Burkholder, 1921; Zaumeyer, 1930). X. axonopodis pv. phaseoli is capable of long-term survival having been recovered from 15 year-old bean seeds (Schuster and Sayre, 1967).

Surface and internal populations of X. axonopodis pv. phaseoli were investigated on and in flower buds, flowers and pods of five susceptible (Pinto UI114, Cranberry Taylor Hort, Charlevoix, Black Magic and C20) and two resistant (Valley and I84100) Phaseolus vulgaris genotypes (Mabagala, 1997). Plants were grown in the field in Tanzania, and artificially inoculated at the seedling stage (18 day old). The pathogen was recovered in high numbers from flower buds, flowers, pods and seed of resistant and susceptible genotypes. Significant differences (P=0.05) in population levels of X. axonopodis pv. phaseoli between stages of reproductive tissue development were observed. Infected seed from resistant genotypes had no visible symptoms. It is suggested that these seeds may play an important role in the epidemiology of common bacterial blight disease because they are difficult to detect and may occur at low frequency in seed lots.

Effect on Seed Quality

Severely infected seed may be shrivelled and show poor germination or produce weakened plants On white-seeded varieties, yellow or brown spots may appear on the seed coat, particularly near the hilum area. On dark-seeded varieties, this discoloration is not visible (Zaumeyer, 1957). Infected seeds may also be symptomless (Yoshii, 1980).

Pathogen Transmission

Seed

Seeds are the primary source of inoculum for X. axonopodis pv. phaseoli. Plants grown from infected seeds frequently bear lesions on the cotyledons or primary nodes. These lesions enlarge and under humid conditions, slimy masses of bacteria accumulate on the leaf surface. These are then spread to healthy plants (Zaumeyer, 1957). Approximately 1000 to 10,000 bacteria per seed is the minimum needed to produce infected plants under field conditions (Weller and Saettler, 1980). A range of tolerances, ranging from 0 seeds infected in samples of 4000-45,000, are used in seed quality programmes in US seed companies to ensure that the pathogen is not transmitted by commercial seed lots (Maddox, 1997).

The most effective survival mechanism for Xap is to inhabit infected bean seed (Cafati & Saettler 1980a, Gilbertson et al., 1990; Arnaud-Santana et al., 1991; Opio et al., 1993), within which bacteria may survive for up to thirty six years (Allen et al., 1998).  Marques et al. (2005) indicated that the survival of seedborne X. axonopodis pv. phaseoli was reduced from 64 to 36-37% incidence during the first 6 months; however, seed stored at –18 and 5 ºC  maintained the contamination rate at 30 and 60 months, and  it was concluded that the optimum temperatures for storing seed is similar to conditions favourable for Xap longevity (Marques et al., 2005). Seed contamination may be internal or external (Saettler 1989; Allen et al., 1998) and even symptomless (Thomas & Graham, 1952; Weller and Saettler, 1980a), which has serious implications for seed certification schemes.  It has, however, been indicated that the development of X. axonopodis pv. phaseoli epidemics are depended on the level of horizontal resistance and climatic conditions rather than the population size of Xap in bean seeds (He, 2010). He (2010) found that Xap had a significant higher percentage of seed to seedling transmission than Xap. Although both variants reduced seedling emergence , Xapf was more severe and the incidence of typical CBB lesions were higher in seedlings infested with Xapf than Xap.

Other sources

Infected crop residues and other susceptible hosts are also sources of inoculum for X. axonopodis pv. phaseoli (Zaumeyer, 1957; Khlaif and Qadous, 1995). Conflicting reports exist on the ability of Xap to survive in infested soil and plant debris (Schuster & Coyne 1976; Saettler et al., 1986;  Gilbertson et al., 1990). Gilbertson et al. (1990) found Xap populations to overwinter in bean debris on no-tillage plots.  Non-pathogenic pectolytic strains of X. campestris were also consistently isolated.  Experiments conducted in the Dominican Republic indicated that Xap survived up to 7 months on infected debris on the soil surface, but not in buried debris after 30 days (Arnaud-Santana et al. 1991).  Xap survival studies conducted over ten years in Michigan indicated that infected crop debris is not the primary inoculum source for CBB (Saettler et al., 1986).  Infected bean debris may be more important as an inoculum source in tropical and sub-tropical than in temperate areas (Gilbertson et al., 1990).

Survival of Xap is greater under dry conditions (Schuster & Coyne 1977a) as bacteria decline rapidly under moist conditions (Allen et al. 1998).  Sabet & Ishag (1969) reported that Xap survived in press-dried bean leaves for more than 18 months in the laboratory, while Gilbertson et al. (1988) found Xap to remain viable in dry-leaf inoculum after 6 years.  The longer survival under laboratory conditions as opposed to that in the field could be attributed the presence of antagonists, such as protozoa, in the soil (Habte & Alexander 1975).

Xap also survives on weeds and other host plants (Cafati & Saettler 1980b; Angeles-Ramos et al., 1991; Opio et al., 1995).  Certain weed species may harbor the pathogen for up to 6 months (Opio et al. 1995).  Angeles-Ramos et al. (1991) isolated epiphytic, pectolytic Xanthomonads from symptomless weeds where pathogenic strains were isolated from within infected fields.  Epiphytic colonies survive on a wide range of plant species in the families Amaranthaceae, Commelinaceae, Compositae, Cruciferae, Gramineae, Oxalidaceae and Portulaceae in addition to various legumes (Allen et al., 1998).  Epiphytic Xap populations are important in the epidemiology of CBB on dry beans (Ishimaru et al., 1991) and are differentially affected in hosts of different genotypes (Cafati & Saettler 1980a).

Seed Treatments

Seed infection by X. axonopodis pv. phaseoli can be external or internal. Both hot water and dry heat have been successful in treating bean seeds for X. axonopodis pv. phaseoli (Gondreau and Samson, 1994). This involves either incubating for 20 minutes in 52°C water or 23-32 hours in 60°C dry air at 45-55% RH. The latter treatment does not appear to affect seed viability. Treatment with an antibiotic such as streptomycin may be used to control external contamination with X. axonopodis pv. phaseoli, and streptomycin in polyethylene glycol may reduce, but not eliminate, internal populations of X. axonopodis pv. phaseoli (Liang et al., 1992).

The application of Rhizobium leguminosarum bv. phaseoli to common bean seeds reduced common bacterial blight severity and imnproved plant growth, both in field and greenhouse conditions (Osdaghi et al., 2011).

Under greenhouse conditions, bean seeds treated with a solution of tolylfluanid (1.20 g/L water) did not show any symptoms of infection until 30 days after sowing, and the overall prevelance of X. axonopodis pv. phaseoli was lower than in plants grown form untreated seeds (Lopes et al., 2008).

No methods are available to eradicate internal seed populations. However, external contamination may be controlled by streptomycin sulphate and sodium hypochlorite (Liang  et al. 1992).  Liang et al. (1992) investigated the potential of osmotic conditioning in reducing internal Xap populations from seeds, using polyethylene glycol (PEG) and glycerol as antibiotic carriers.  They found that tetracycline and chlorotetracycline in PEG solutions effectively reduced Xap, but were phytotoxic.  PEG solutions containing streptomycin reduced, but did not eradicate internal bacterial populations from naturally infected seeds with few phytotoxic effects. Tolylfluanid and Streptocycline were also reported as effective seed treatments (He, 2010).

Streptomycin is rapidly absorbed into bean stems and translocated to leaves but there is no indication that antibiotics are translocated downward through stems, trifoliate leaves or peduncle into the pod (Mitchell  et al. 1954).  Antibiotics should not be applied to leaves as resistant mutants may develop (Saettler 1989).  Development of resistance to chemicals (Romeiro et al. 1998), costs involved and efficacy limit use of chemical control which may be feasible under certain circumstances, such as seed production or as a component of an integrated control strategy (Allen et al., 1998).

Seed Health Tests

Selective medium (Mohan and Schaad, 1987; Higley et al., 1993)

- Soak seeds for 20 h in a solution of 0.85% NaCl and 0.01% Tween 20 (SST) at 5°C.
- Aliquots of the soak solution are spread on a semi-selective medium (M-SSM) developed by (Mabagala and Saettler, (1992).
- Bacterial colonies that develop are isolated and tested for pathogenicity by inoculation onto greenhouse-grown bean plants (cv. Yelloweye).

As indicated by Maddox (1997) samples sizes can vary greatly (4000-45,000 seeds) in routine seed quality tests for this pathogen.

Dome test (Venette et al., 1987)

In this test, bean seeds are soaked in water. The soaked bean infusion is then vacuum-infiltrated into pregerminated seeds of the same lot. Symptoms are then observed after seedlings are grown out at high humidity.

Phage (Kahveci and Maden, 1994)

High degrees of specificity and reliability for detection of seedborne X. axonopodis pv. phaseoli was claimed for 2 of 16 bacteriophages obtained from X. axonopodis pv. phaseoli.

Serology (Wong, 1991)

Immunoflourescence and ELISA methods have been described.

DNA hybridization (Audy et al., 1996)

X. axonopodis pv. phaseoli can be detected in bean seeds after DNA extraction of intact or crushed seed followed by a PCR assay using pathovar-specific primers (Audy et al., 1996). Another PCR assay has been developed that is specific to X. axonopodis pv. phaseoli var. fuscans and thus will not detect X. axonopodis pv. phaseoli (Toth et al., 1998).

Nondestructive assay (Higley et al., 1993)

Various non-destructive methods detected X. axonopodis pv. phaseoli in bean seeds.

Real-time PCR (He, 2010)

Real-time PCR offers a sensitive, reliable and fast approach to diagnosis of Xap and Xapf in seeds (He, 2010).  Assay specificity has been tested against DNA of several Xanthomonas species and pathovars of X. axonopodis. None of the closely related Xanthomonas strains were amplified using this PCR assay.

Many different diagnostic methods have been developed for X. axonopodis pv. phaseoli and X. axonopodis pv. phaseoli var. fuscans (Xapf). Xap and Xapf can be easily isolated from CBB symptoms on leaves and pods using general isolation media (Schaad & Stall 1988).  On media such as sucrose peptone agar (SPA), colonies are circular, smooth and mucoid with a yellow pigment referred to as xanthomonadin.  Intensity of this yellow colour varies with medium used (Moffet & Croft 1983).  Isolation media containing tyrosine differentiates between Xap and Xapf in that the latter produces a brown diffusible pigment (Basu & Wallen 1967).  Goodwin & Sopher (1994) found this pigment to be produced due to secretion and subsequent oxidation of homogentisic acid rather than tyrosine activity.

Selective media are more effective for isolating specific bacteria from diseased material when selective at species level (Claflin, et al., 1987).  Examples of semi-selective media for X. axonopodis pv. phaseoli are MXP (Claflin et al., 1987), milk-tween agar (Goszczynska and Serfontein, 1998) and M-SSM (Mabagala and Saettler, 1992). Remeeus and Sheppard (2006) recommended the use of semi-selective media MT and XCP1 for the detection of Xanthomonas axonopodis pv. phaseoli. They indicated that although MT was not as effective than XCP1 a larger number of suspected and confirmed colonies of Xanthomonas axonopodis pv. phaseoli were found on MT versus XCP1.  A number of semi-selective media are, however, available to isolate Xap and Xapf (Kado & Heskett 1970, Schaad & White 1974, Trujillo & Saettler 1979, Claflin et al. 1987,  Dhanvantari & Brown 1993, Jackson & Moser 1994).

Apart from using selective media, techniques such as bacteriophage typing (Katznelson et al. 1954, Sutton & Wallen 1967), serology testing (Trujillo & Saettler 1979), host inoculation (Saettler 1971), ELISA (Wong 1991) and immunofluorescent staining  (Malin et al. 1983), can be used to detect and identify Xap and Xapf.  These techniques are time consuming and labourious.  More sensitive, rapid and specific detection of the pathogen is often needed.  This is particularly important when identification is complicated by epiphytic Xap strains (Gilbertson et al., 1989, Audey et al., 1994) that may confuse seed certification (Wong 1991, Audey et al. 1994).

Starch is frequently included in the medium to help detect X. axonopodis pv. phaseoli because it hydrolyses starch, an uncommon trait among bacteria. Various diagnostic physiological tests are available for use with pure cultures but a pathogenicity test is required for detection at the pathovar level (Sheppard et al., 1989). Detection with phages specific to X. axonopodis pv. phaseoli have also been attempted, but not all strains are susceptible to a particular phage and phages are usually not truly species-specific (Sheppard et al., 1989).

Immunosassays are available for X. axonopodis pv. phaseoli but most have not been sufficiently specific for accurate diagnosis. However, monoclonal antibodies are now available for X. axonopodis pv. phaseoli and have been used in immunofluorescence microscopy and an ELISA to detect it in seed (Wong, 1991). This monoclonal antibody appears to be highly specific to X. axonopodis pv. phaseoli and only cross-reacts with X. axonopodis pv. malvacearum (Wong, 1990).

Specific diagnosis of X. axonopodis pv. phaseoli was achieved with a DNA hybridization probe (Gilbertson et al., 1989). Based on this probe, another highly specific PCR probe, for Xap detection, was developed to detect as few as 10 colony forming units (CFU), using ethidium bromide-stained agarose gel (Audey et al. 1994).  Audey et al. (1996) developed a rapid, sensitive PCR assay for detection of seedborne Xap in large bean seed samples containing as few as one infected in 10 000 healthy seeds.  Birch et al. (1997) used RAPD-PCR to differentiate between Xap and Xapf.  Toth et al. (1998) used  primers which amplified a DNA fragment from all Xapf-isolates used, while no amplification products were obtained from Xap-isolates.  These primers, therefore, provide a rapid, improved method to differentiate between these two variants. More recent techniques used to differentiate Xap and Xapf include restriction fragment length polymorphism (RFLP) analyses (Chan et al., 1990; Gilbertson et al., 1991, Lazo et al., 1987), DNA-DNA hybridization (Hildebrand et al., 1990), pulse field gel electrophoresis (Chan et al., 1990) and rep-PCR (López et al., 2006; Mahuku, 2006; Mkandawire et al., 2004).

The pathogen can be readily isolated from symptomatic tissue (Dreo et al., 2003). A comparison of various extraction processes is given in He and Munkvold, 2012).

The method of identification issued by the International Seed Federation in 2006 is successfully used in Balaz et al. (2008). X. axonopodis pv. phaseoli was isolated from bean seeds and isolated on a semiselective media. ELISA and PCR can then be used to confirm the identity of resulting isolates. Milk Tween Agar (MTA) and Modifies Sucrose Peptone Agar (MSPA) are both suitable for isolation of P. savastanoi pv. phaseolicola.

The development and use of a PCR-based method for detection of X. axonopodis pv. phaseoli in pomegranate is described in Mondal et al. (2012).

Seed Health Tests

A sample, usually 1000 to 10,000 seeds, is washed in running water and then placed in a container to which a solution of phosphate-buffered saline or liquid semi-selective media is added. The samples are then incubated at room temperature for 2-3 hours. Longer incubation times can increase the number of saprophytic bacteria and reduce the number of aerobic xanthomonads. The solution, known as the seed-soak wash, is diluted and plated onto various culture media. The bacteria are then identified by physiological traits, phage tests, pathogenicity tests and/or immunoassays (Sheppard et al., 1989). The seed-soak wash can also be directly injected into susceptible plants or vacuum-infiltrated into pregerminated bean seeds, which are then observed for typical common blight symptoms (Venette et al., 1987; Sheppard et al., 1989).

A comparison of extraction methods showed that crushing seed in phosphate-buffered saline was more effective in releasing bacteria than soaking intact seeds (Maringoni et al., 1998). X. axonopodis pv. phaseoli can also be detected in seed after DNA extraction of intact or crushed seed followed by a PCR assay using specific primers (Audy et al., 1996). Results from studies conducted by He (2010) to assess the influence of different extraction steps on the sensitivity of Xap indicated that vacuum extraction and centrifugation of seed extracts increased sensitivity, and the highest sensitivity was obtained with the 3-hour vacuum extraction at room temperature followed by centrifugation.

For infected plant tissue other than seed, the material should be washed thoroughly in running water and dried. Surface sterilization may be necessary as a general hygiene precaution to avoid surface contaminants. The margin of a lesion is excised with a sterile blade and mixed with a small amount of sterile water or phosphate-buffered saline to allow the bacteria to diffuse out into the solution for 15-30 minutes. A sterile loop is then used to streak the solution onto agar media (Schaad and Stall, 1988).

Due to the variable regulations around (de)registration of pesticides, your national list of registered pesticides or relevant authority should be consulted to determine which products are legally allowed for use in your country when considering chemical control. Pesticides should always be used in a lawful manner, consistent with the product's label.