Curtobacterium flaccumfaciens pv. flaccumfaciens
(bacterial wilt of dry beans)

Curtobacterium flaccumfaciens pv. flaccumfaciens, the causal agent of bacterial wilt of dry beans, is an economically important pathogen of edible legumes (i.e., common bean, soyabean, mungbean and cowpea) around the world. The disease occurs in many countries with a particular importance in regions characterized by dry and hot summers with extended drought. As a seedborne pathogen, C. flaccumfaciens pv. flaccumfaciens is included in the A2 (high risk) list of quarantine pathogens by the European and Mediterranean Plant Protection Organization (EPPO); hence, it is under strict quarantine control and zero tolerance in the dry bean industry in several countries. Bacterial wilt was first reported on common bean in South Dakota, USA, in 1922 and then spread into South America, Canada, Australia, Europe and more recently to western Asia (Iran) and Africa. Identification of the pathogen can be difficult owing to the presence of five different colony colour variants (i.e., yellow, orange, pink, purple and red) on culture media.

Bacterial wilt was first described in South Dakota, USA, in 1922 (Hedges, 1922). In addition to the USA, the disease was reported in Canada and Mexico in 1955 (Patrick, 1955; Yerkes and Crispin, 1956). However, its presence in Mexico has not been confirmed (EPPO/CABI, 1997). The disease was restricted to North America until being observed on mungbean and cowpea plants in Australia in the late 1980s (Wood and Easdown, 1990). The disease has also continued to spread southward into South America, reaching Colombia in 1982 (Torres et al., 1982) and Venezuela in 1990 (EPPO, 2011). Then, Maringoni and Rosa (1997) reported the disease on common bean plants in São Paulo State, Brazil in 1995. Since the beginning of the 21st Century, bacterial wilt has sporadically been reported in some European countries (EPPO, 2011). For instance, the disease was found affecting common bean in south-eastern Spain in 2005 (González et al., 2005). Subsequently, C. flaccumfaciens pv. flaccumfaciens was isolated from soyabean plants in Germany in 2012 (Sammer and Reiher, 2012). The disease was also reported, but not substantiated, in two distinct areas of Russia (EPPO, 2011). The pathogen was not officially reported in Asia until 2013 when a widespread occurrence of the disease was found in Iran in both common bean and cowpea fields from different provinces (Osdaghi et al., 2015a, b). Unofficial occurrences of the pathogen have also been recorded in Albania, Belgium, Bulgaria, France, Greece, Hungary, Romania, Switzerland, Tunisia, Turkey and former Yugoslavia (EPPO, 1994; Ishimaru et al., 2005). Although the pathogen has previously been observed infecting common bean in Africa (i.e., Kenya, Mauritius, South Africa and Tunisia; EPPO, 2011), it was not officially documented from Africa until 2019 when it was reported on soyabean plants in Zambia (Pawlowski and Hartman, 2019).

Risk Criteria Category

Economic Importance Moderate to high depending on the region and host crop
Distribution North and South America, Western Asia, Australia
Seedborne Incidence Moderate
Seed Transmitted Yes
Seed Treatment None

Overall Risk Moderate to high

Notes on phytosanitary risk

Although C. flaccumfaciens pv. flaccumfaciens is widespread geographically, imported seed is often subject to declaration of disease status. Exporters of common bean, soyabean and mungbean are often required to certify their exports free from disease to enable entry into other countries. 

Apart from common bean (Phaseolus vulgaris), the natural host list of C. flaccumfaciens pv. flaccumfaciens includes adzuki bean (Vigna angularis), black gram (Vigna mungo), cowpea (Vigna unguiculata), hyacinth bean (Lablab purpureus), lima bean (Phaseolus lunatus), mungbean (Vigna radiata), pea (Pisum sativum), scarlet runner bean (Phaseolus coccineus), soyabean (Glycine max), yardlong bean (Vigna unguiculata subsp. sesquipedalis) and Zornia glabra (Dunleavy, 1983; Chavarro et al., 1985; Ishimaru et al., 2005; Harveson and Vidaver, 2007; Osdaghi, 2014; Osdaghi et al., 2015b).

C. flaccumfaciens pv. flaccumfaciens is seedborne, with poor survival in soil. It overwinters in plant debris and in weeds. Contaminated seeds provide the initial source of infection, which becomes systemic in the xylem. Mechanical injury of roots by infections from the root-knot nematode Meloidogyne incognita, was shown to provide a mechanism for introducing the pathogen into host plants (Schuster 1959). Bacteria may also enter through wounds on roots and above-ground plant parts and, in contrast to other common bacterial pathogens, infrequently through stomata. Secondary infections occur through wounds following rain and hail storms. Disease is favoured by temperatures above 30°C. Moisture stress induces wilting in common bean and Zornia spp.

Survival of C. flaccumfaciens pv. flaccumfaciens in intact soil under field conditions varies between 34 and 80 days as a function of temperature and moisture (Gonçalves et al., 2018). Infested common bean debris and straw are important in C. flaccumfaciens pv. flaccumfaciens overwintering. Bacteria in infested dry straw on the soil surface survive far better (up to 22 months) than in debris buried 20 cm in the soil (Schuster and Coyne, 1974). Non-leguminous plant species were shown to harbour the pathogen for overwintering (Schuster, 1970; Saettler, 1991). Gonçalves et al. (2017) showed that under Brazilian field conditions, C. flaccumfaciens pv. flaccumfaciens colonizes barley, black oat, oilseed rape, ryegrass, wheat and white oat where these plant species are cultivated during winter in rotation with common bean. It is recommended that common bean cultivation in succession with barley, black oat, oilseed rape, ryegrass, wheat and white oat must be avoided in areas with a history of the disease. The pathogen endophytically colonizes stems of wheat plants inoculated by wounding or spraying techniques (Silva Júnior et al., 2012). Harveson et al. (2015) isolated bean-pathogenic orange and yellow strains of C. flaccumfaciens pv. flaccumfaciens from black chaff-infected wheat plants. Black chaff is a bacterial disease on small grain cereals caused by different pathovars of Xanthomonas translucens. Dry bean-pathogenic strains of C. flaccumfaciens pv. flaccumfaciens were also isolated from symptomless aubergine, pepper and tomato plants in Iran (Osdaghi et al., 2018a).

Incidence

Contaminated seeds are considered to be the main method of introducing bacterial wilt inoculum into areas with no history of the disease (Zaumeyer and Thomas, 1957; Saettler and Perry, 1972). The level of seed transmission was reported to range from six to 70 percent in different assays, however, transmissibility of the pathogen via seeds depends on the host plant and time of infection (Wood and Easdown, 1990; Camara et al., 2009). Up to 10% of commercial mungbean seeds were found to be infected by C. flaccumfaciens pv. flaccumfaciens after surface sterilization with NaOCl (Tripepi and George, 1991). The pathogen has been shown to be present in soyabean seeds collected in 14 US states and in Ontario, Canada (Dunleavy, 1988). It is also seedborne in common bean (Schuster and Smith, 1983) and cowpea (Vigna unguiculata) seeds (Arcila and Trujillo, 1990). Masses of C. flaccumfaciens pv. flaccumfaciens were localized in testa cells of seeds of Zornia spp. (Chavarro et al., 1985).

Effect on Seed Quality

C. flaccumfaciens pv. flaccumfaciens is included in the A2 (high risk) list of quarantine pathogens by EPPO; hence, it is under strict quarantine control and zero tolerance in dry bean industry in several countries (EPPO, 2011). Economic losses caused by bacterial wilt disease are attributed not only to a substantial decrease in the net crop yield (i.e., number of pods per plants and number of mature seeds per pod) but also to a significant decrease in marketability of the product where visual appearance, shape and size of the seeds are determinative factors in pricing (Huang et al., 2009). C. flaccumfaciens pv. flaccumfaciens causes yellow or purple discoloration on common bean seeds but does not affect germination (Schuster and Sayre, 1967). However, the sprouting quality of mungbean can be affected (Conde and Diatloff, 1991) and seed infection by C. flaccumfaciens resulted in low germination of seeds of Zornia spp. (Chavarro et al., 1985). In visual inspections of dry bean fields it should be noted that seeds may become infected even when pods look healthy (Harveson et al., 2006). When bacterial wilt occurs on older plants, disease and symptom development proceed more slowly and are less severe. The percentage of common bean seeds possessing bacterial wilt symptoms in a given field (i.e., yellow, orange, pink or purple discoloration of the seed coat) was reported to vary between 0 and 25% in seed-producing fields in Canada and research plots in Nebraska, USA (Huang et al., 2003, 2004; Harveson et al., 2006, 2015).

Seed Transmission

C. flaccumfaciens pv. flaccumfaciens is primarily transmitted by contaminated seeds that carry the pathogen both externally and internally. It can also be found on overwintering bean straw (Schuster and Smith, 1983). Artificially inoculated mungbean seeds gave 6-12% transmission of C. flaccumfaciens pv. flaccumfaciens (Wood and Easdown, 1990). The bacteria may also be transmitted to mungbean via Ipomoea species: infected plants act as alternative weed hosts (Conde and Diatloff, 1991). Localized spread by irrigation water may also occur (Hedges, 1926). The rate of transmission in common bean seeds is reported to be higher than in other hosts of the pathogen. For instance, susceptible and resistant soyabean cultivars showed 0-1% and zero seed transmission, respectively; while up to 70% seed transmission was reported in susceptible common bean cultivars in greenhouse experiments (Camara et al., 2009; Soares et al., 2018). Bacterial tan spot lesions developed on soyabean seedlings grown in sterile soil in growth chambers from surface-sterilized seeds. Transmission rates averaged 13.5% and 7.0% for susceptible cultivars grown at 25°C and 30°C, respectively. No lesions were detected on seedlings grown at 10, 15, 20 or 35°C (Dunleavy, 1986).

Seed Treatments

Seed inspections during intra- and international transportation of dry beans should be employed to avoid the distribution of the pathogen to new areas with no history of the disease (EPPO, 2011). There are no effective seed treatments against C. flaccumfaciens pv. flaccumfaciens. Copper and antibiotics were ineffective (Tripepi and George, 1991) as were experimental heat treatments at 52°C for 20 h and 85°C for 5 h (Zaumeyer and Thomas, 1957).

Harding et al. (2007) concluded that resistance to chemicals is attributed to the capacity of the bacterial wilt pathogen in biofilm production. Recently, Harveson (2019) reported the results of a 7-year (2010-2016) field study (13 site years) investigating the performance of newly available antimicrobial chemicals for managing the four major bacterial diseases of dry beans (i.e., common blight, halo blight, brown spot and wilt). These novel products were compared with two conventional copper‐based products, copper hydroxide and copper sulfate, in Nebraska, USA, and several showed great promise for reducing losses due to these diseases. Treatments with hydrogen peroxide and peroxyacetic acid and ecoAgra 300 (plant-based fatty acids) consistently produced higher seed yields than the other treatments compared to the controls while not reducing disease incidence. These products do not appear to be curative, but act as protectants by slowing or prohibiting disease progress. Better results in all seasons were achieved from severely affected fields, than those from fields with low levels of disease (Harveson, 2019).

Soares and Maringoni (2002) evaluated the use of acibenzolar-S-methyl and its effect on the germination and vigour of common bean seeds and the induction of resistance to bacterial wilt. Results indicated that the seed treatment was phytotoxic to seeds, reducing the number of normal plants that developed in germination and vigour tests. Acibenzolar-S-methyl did not efficiently induce resistance to bacterial wilt when applied either as a seed treatment or as a spray. Ryley et al. (2010) suggested that the use of clean seed from a certified source and resistant cultivars are equally important in the management of bacterial wilt (tan spot) on mungbean in Australia, while crop rotation played a minor role.

Antibiotic seed treatment can reduce surface contamination of seeds. For instance, slurry seed treatments using Streptomycin Agri-Strep 500 were shown to be effective to combat C. flaccumfaciens pv. flaccumfaciens infection on common bean seeds (Schwartz, 2007). Soaking the seeds in Agrimaicin 500 solution, containing copper sulphate and oxytetracycline, eliminated the bacterium from naturally infected seeds; however, it was not effective on the artificially inoculated seeds using 10⁸ c.f.u./ml suspension of the pathogen (Estefani et al., 2007).

Seed Health Tests

Grow-out test (Dunleavy, 1986; USDA National Seed Health System Standard Method, www.seedhealth.org).

1. Surface-sterilize the suspected dry bean seeds and plant in sterile soil.
2. Grow seeds at 30°C under growth-chamber conditions until characteristic leaf symptoms are visible. Refer to EPPO (2011) for further details.

Serological tests (Calzolari et al., 1987)

Although a number of serological techniques were developed for detection of C. flaccumfaciens pv. flaccumfaciens, they are unable to differentiate non-pathogenic lineages of C. flaccumfaciens from the pathogenic members (Calzolari et al., 1987; Diatloff et al., 1993; McDonald and Wong, 2000). Immunofluorescence staining was successful in the detection of C. flaccumfaciens pv. flaccumfaciens to a concentration of 1.23 × 10⁷, which corresponded to 1.82 × 10⁴ fluorescent cells per ml of soaked seed leachate (Calzolari et al., 1987). Immunofluorescence tests using a polyclonal antibody produced against C. flaccumfaciens pv. flaccumfaciens strain NCPPB 559 lacked adequate sensitivity and did not react with all tested C. flaccumfaciens pv. flaccumfaciens strains (Calzolari et al., 1987; McDonald and Wong, 2000), while a monoclonal antibody developed by Diatloff et al. (1993) was able to detect the pathogen from symptomless mungbean plants.

Two methods have been compared for detecting C. flaccumfaciens pv. flaccumfaciens in commercial common bean seed lots. Experimentally contaminated seeds were homogenized and a standard extraction and concentration procedure used. The final pellets were used for immunofluorescence staining and indirect isolation through seedlings. The staining sensitivity threshold was 12,300,000, corresponding to 18,200 fluorescent cells per millilitre of final concentrate. The indirect isolation sensitivity threshold was 1,230,000, producing 43% symptomatic plants and 53% positive re-isolation.

Pathovar Specific PCR Primers

Two PCR tests for the detection of C. flaccumfaciens pv. flaccumfaciens in bean seeds are described in the literature (Guimaraes et al., 2001; Tegli et al., 2002). A highly sensitive and rapid PCR assay for the detection of C. flaccumfaciens pv. flaccumfaciens in common bean seeds was developed by Tegli et al. (2002). A pair of PCR primers (CffFOR2-CffREV4), targeting the sequence of a cloned DNA fragment of 550 bp amplified in repetitive-sequence-based-PCR (Rep-PCR) experiments, were designed and shown to specifically amplify a 306-bp DNA fragment using C. flaccumfaciens pv. flaccumfaciens DNA as template. Moreover, this PCR protocol successfully detected C. flaccumfaciens pv. flaccumfaciens in naturally infected bean seeds in 36 h. Using these primers in combination with Bio-PCR and centrifugally concentrated seed extract allows the detection of one artificially contaminated common bean seed among 999 healthy seeds (Deuner et al., 2012). The primer pair CffFOR2/CffREV4 did not direct the amplification of the expected 306 bp fragment in various non-pathogenic C. flaccumfaciens strains isolated from tomato and aubergine (Osdaghi et al., 2018a). Tegli et al. (2017) provided a detail-oriented step-by-step protocol for detection of C. flaccumfaciens pv. flaccumfaciens in dry bean seeds using both general and semi-selective culture media as well as conventional PCR.

General

• Laboratory use
• Research model

The bacterial wilt pathogen can readily be isolated from all above-ground parts of dry beans, but leaves, stems and seeds are the most suitable. Isolation of C. flaccumfaciens pv. flaccumfaciens from either seed lots or symptomatic plant tissues can be performed by cutting the marginal tissues of the visible lesions on leaves and grounding them in sterile distilled water (SDW). Spread a loopful of the resulting suspension on NBY (5 g glucose, 2 g K₂HPO₄, 0.5 g KH₂PO₄, 0.25 g MgSO₄, 8 g nutrient broth, 2 g yeast extract and 15 g agar dissolved in 1 L SDW) or YPGA (10 g glucose, 5 g peptone, 5 g yeast extract and 15 g agar dissolved in 1 L SDW) media (EPPO, 1994, 2011). The bacterium grows also on a broad range of media, including Kelman’s medium, nutrient agar, MSC medium and King’s medium B (Tegli et al., 2017). Several methods for isolation can be employed. Margins of leaf lesions can be abraded or punctured with a dissecting needle, and streaked onto medium plates (Harveson et al., 2006; Harveson et al., 2015). Another technique involves squeezing the sap out of petioles attached to symptomatic tissues and blotting onto medium, followed by streaking with a sterilized inoculating loop (Harveson, 2013). For dried leaf samples, leaf tissues can be ground in SDW, shaken for 1-2 h to release the bacterial cells into the water, and a loopful of the resulting suspension can then be spread onto YPGA or NBY media. Another method of isolation can be utilized by placing dried tissues in humidity chambers for 12-24 hours. If present, bacteria will ooze out of necrotic tissues and can be streaked onto media (Harveson et al., 2015). Finally, the bacterium can also be isolated from infected, discoloured or even symptomless seeds. Discoloured seeds are soaked overnight in SDW and the eluent is streaked onto plates (Harveson and Schwartz, 2007). After streaking on the media surface, fluidal colony growth will be observed on the culture media after incubation at 25-27°C for 48-72 h.

To confirm whether a suspected plant is infected with the bacterial wilt pathogen, excise a piece of discoloured vascular tissue, or a discoloured seed, or the necrotic margin of the infected leaf tissue and comminute in sterile water. Streak bacterial suspension onto a glass slide and carry out Gram-staining. If Gram-positive, coryneform bacteria are detected in the suspension in large numbers and the plants show typical symptoms of wilt or spotting, a presumptive diagnosis can be made. For further confirmation, a loopful of the resulting suspension can be streaked onto yeast extract (7 g/litre) peptone (7 g/litre) glucose (7 g/litre) agar (18 g/litre) (YPGA) medium. The plates should be incubated at 27-30°C for 4-5 days. The appearance of yellow, orange or red-pigmented bacterial colonies is an approval of infection of the plant in question. Diagnosis as C. flaccumfaciens pv. flaccumfaciens can be confirmed by either specific PCR primers as described by Tegli et al. (2002) or stem-prick inoculation with bacteria into common bean (Phaseolus vulgaris), which is a suitable test plant for the various host isolates (for a detailed description, see Osdaghi et al. (2020) and Tegli et al. (2017)).

In growing crops, C. flaccumfaciens pv. flaccumfaciens can be detected as leaf chlorosis and large, tan-coloured spotting with or without wilting. Infected common bean seeds may be discoloured yellow, orange or purple, but discoloration does not occur on soyabean, cowpea or mungbean, which are symptomless carriers.

Bacterial wilt symptoms may be confused with those caused by the common bacterial blight pathogen, Xanthomonas axonopodis pv. phaseoli in areas where both pathogens occur in the same field (Harveson et al., 2011). Bacterial wilt lesions occur between veins, often accompanied by wilting and death of severely infected plants. Whereas, wilting and plant death are less likely to occur in common bacterial blight and other bacterial diseases, i.e., halo blight and brown spot caused by Pseudomonas savastanoi pv. phaseolicola and P. syringae pv. syringae, respectively (Schwartz et al., 2005; Harveson and Schwartz, 2007), although neither of these pathogens is systemic. Isolation in a laboratory would be necessary to distinguish these pathogens.

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.

No effective chemical control is available against C. flaccumfaciens pv. flaccumfaciens, although secondary spread can be retarded by bactericidal sprays of copper fungicide. Weeds, volunteer plants and plant debris that act as reservoirs of infection should be eliminated.

Heat treatment does not eliminate the pathogen from seeds; there are no established seed treatments. Development of resistant cultivars is the most practical method for managing bacterial wilt disease in terms of cost effectiveness and durability over time. Common bean cultivars and varieties differ in their reaction to the pathogen, and resistant or tolerant varieties should be planted, if available, in the areas where the disease is established (Hsieh et al., 2005; Urrea and Harveson, 2014). Furthermore, considering the differences among C. flaccumfaciens pv. flaccumfaciens colony variants in their virulence and aggressiveness, a given cultivar/line should be evaluated for its resistance against different variants of the pathogen (Huang et al., 2007; Conner et al., 2008; Urrea and Harveson, 2014).

As for the biological control methods, Corrêa et al. (2014) applied a combination of Bacillus cereus and Pseudomonas fluorescens strains to reduce the severity of bacterial wilt by seed inoculation using the antagonistic bacterial suspension. Bacterial wilt reduction ranged from 42 to 76% (Martins et al., 2013). Environmental temperature (20 vs. 30°C) interferes with the colonization of plants with the pathogen and antagonist, while the suppression of bacterial wilt using the B. cereus strain ALB629 was similar in the two temperatures (Martins et al., 2014). Furthermore, soaking common bean seeds in a suspension (3×10⁸ c.f.u./ml) of Pantoea agglomerans resulted in thorough endophytic colonization of the entire bean seedling from root to apical stem 7 days post inoculation and resulted in the reduction of bacterial wilt severity in greenhouse conditions (Hsieh et al., 2005). The application of P. agglomerans as a soil drench 24 h post planting was effective in suppressing bacterial wilt, but not as effective as seed treatment (Hsieh et al., 2005). Furthermore, seed treatment using Rhizobium leguminosarum reduced bacterial wilt and common bacterial blight diseases in greenhouse conditions (Huang et al., 2007b). Bacteriophages have also been proposed for the control of the bacterial wilt pathogen but no detailed information is available in this regard (Klement 1957).

29/08/20 Reviewed by:

Ebrahim Osdaghi, Department of Plant Protection, University of Tehran, Karaj 31587-77871, Iran.