Pseudomonas cichorii
(bacterial blight of endive)

P. cichorii is probably ubiquitous. It causes serious diseases in all regions characterized by mild temperatures and high humidity or in protected environments. It is widespread in France, Germany, Italy, Russia, UK, Japan, Taiwan, South Africa, Tanzania, Barbados, Brazil, Cuba and New Zealand. In the USA, it occurs most frequently in Florida.

P. cichorii is included in the EPPO list of pathogens associated with the import of tomato fruit (EPPO 2015).

P. cichorii has a wide host range and can incite disease in a large number of plant species. Under favourable environmental conditions, the bacterium can cause severe disease in almost all the crops in which it has been described. Some crops have been more widely investigated because of their economic importance and/or the frequent or yearly occurrence of the disease. These hosts include vegetables such as borage (Borago officinalis) (Cambra et al., 2004), celery (Pernezny et al., 1994), endive and lettuce (Grogan et al., 1977); flowering ornamentals such as chrysanthemum (Chrysanthemum x morifolium) (Strider and Jones, 1977; Jones et al., 1983), African daisy (Gerbera jamesonii) (Miller and Knauss, 1973), geranium (Pelargonium spp.) (Engelhard et al., 1983); and foliage ornamentals belonging to the Araceae and Araliaceae (Chase, 1987a). Other hosts from Bradbury (1986) and more recent reports include okra (Mariano et al., 1994), garlic (Stefanova et al., 1992), Peruvian carrot (Arracacia xanthorrhiza) (Beriam et al., 1998), Chinese cabbage (Brassica pekinensis) (Sun et al., 1993), safflower (Carthamus tinctorius) and cornflower (Centaurea cyanus) (Young et al., 1987), Ficus lyrata (Chase 1987b), soyabean (Nishiyama et al., 1986), Hedera helix (Hassanzadeh, 1993), Helianthus (Robbs and Almeida, 1981), Hibiscus helatus (Rodriguez and Hinojosa, 1986), H. rosa-sinensis (Chase, 1986), Hyoscyamus muticus (Sattar et al., 1986), Lagascea mollis (Cruz and Stefanova (1992), Lobelia ricardii (Putnam, 1999), Mentha arvensis (Maia et al., 1996), Musa (Choi et al., 1988), Ocimum basilicum (Miller and Burgess, 1987), Thai basil (Ocimum basilicum var. thyrsiflora) (Luiz et al., 2018), Panax pseudoginseng (Gvozdyak and Pendus, 1988), Phaseolus vulgaris (Cruz and Stefanova, 1992), Plectranthus australis (Miller, 1991), Ranunculus acris (Young et al., 1987), Rhododendron catawbiense (Uddin and McCarter, 1996), turmeric (Curcuma longa) (Maringoni et al., 2003), muskmelon (Cucumis melo), watermelon (Citrullus vulgaris) (Obradovic and Arsenijevic, 2002) and Xanthosoma brasilense (Romeiro et al., 1988).

The experimental host range includes an even larger number of hosts from various botanical families (Bradbury, 1986).

Until recently, the genomic data of P. cichorii has rarely been available (Ramkumar et al. 2015), hence, most of the pathogen-plant interaction mechanisms remain undetermined. P. cichorii has a broad host range and low host specificity. Under favourable environmental conditions, this constitutes a high potential for the development of epidemics. Infection occurs through stomata or wounds. Wounding is not required for infection of most hosts when plants are subject to high levels of free moisture, such as under misting, while inoculum of P. cichorii is present. Under environmental conditions that favour the pathogen, lettuce may be infected through epidermal hairs (Shirata et al., 1982). Wounds caused by the feeding and oviposition of Lyriomiza trifolii provide entry sites for P. cichorii in chrysanthemum (Matteoni and Broadbent, 1988). The bacterium multiplies in the intercellular spaces of the epidermis and then colonizes the intercellular spaces of the mesophyll (Hikichi et al., 1996a). Browning of the tissue develops after bacterial multiplication. Extracts of a phytotoxin, isolated from several P. cichorii strains and partially characterized (Hu et al., 1998), has been shown to induce lesions when injected into lettuce tissues, similar to those induced by the bacterium, and may be responsible for disease symptoms (Shirakawa et al., 1998).

Huang et al. (2015) showed that P. cichorii strain SF1-54 causing lettuce midrib rot disease, produces seven bioactive compounds with biosurfactant properties among which two phytotoxic compounds 'cichopeptin A and B' contribute to virulence of the pathogen on lettuce. Although the cipA-mutant strain (lacking cichopeptin A and B) exhibited significantly less virulence and rotten midribs than the wild type strain, the two strains showed the same level of leaf multiplication on lettuce (Huang et al., 2015). Furthermore, it has been shown that the gene hrpW is involved in the virulence of P. cichorii and it contributes in the induction of hypersensitivity reaction in tobacco (Kajihara et al., 2012). On the other hand, it has been shown that induction of apoptotic cell death by P. cichorii within the infected lettuce tissues initiates the development of rotting symptoms on the plant (Kiba et al., 2006).

Periods of high humidity and leaf wetness are necessary for infection and disease development. The longer the period of moisture the greater is the incidence of the disease (Jones et al., 1984). Lesions cease to expand in low moisture conditions. Thus, plants grown in polyethylene-covered greenhouses or tunnels, in nurseries as well as plants subjected to rainfall or sprinkle-irrigation tend to develop the most severe symptoms. The greenhouse is an ideal environment for disease development because of the dense plant canopy and high humidity.

Disease develops over a wide temperature range, while the optimum temperature is 20-28°C. At higher temperatures, lesion expansion declines and ceases at temperatures above 36°C (Jones et al., 1984).

Experiments on the effect of host nutrition showed that high fertilizer application, stimulating rapid and succulent plant growth, results in an increased number of lesions after inoculation with P. cichorii (Jones et al., 1984). Contrasting results have been obtained on dwarf schefflera (Schefflera arboricola) (Chase, 1987a). Different host species (chrysanthemum, lettuce and coffee) are known to carry epiphytic populations of P. cichorii. On lettuce, the bacterial population increased on the outer and head leaves in the early and middle stages of head formation, respectively (Hikichi et al., 1996b). The bacterium may also be present in apparently healthy chrysanthemum buds (Jones et al., 1990). It has been shown that infections occur only when the pathogen reaches populations on leaves that are greater than 100,000 CFU/g of fresh weight (Hikichi, 1996b). Disease incidence is also correlated with the population level of the bacterium on lettuce leaves (Hikichi et al., 1996b).

P. cichorii can survive in soil for short periods, while it survives for up to 6 months in buried infected debris, but this is not the case under conditions where they are subject to rapid decay (Ohata et al., 1982; Bazzi et al., 1984). The bacterium is frequently associated with other fluorescent pseudomonads in soils and in the rhizosphere of vegetables and weeds (Grogan et al., 1977). It has also been isolated from dried, diseased lettuce leaves that had overwintered for 4 months in a vinyl house, from rotted leaves of Senecio vulgaris and Capsella bursa-pastoris growing in lettuce fields affected by the disease and from their root surfaces, and from those of lettuce and Artemisia princeps (Ohata et al., 1982). P. cichorii which survives within an infected field is able to colonize new plants in the following year. Populations of the pathogen may be present but the disease does not always occur unless environmental conditions favour bacterial multiplication in the host. The bacterium has been isolated from artificially inoculated seeds stored at 23 and 5°C after 50 and 93 days, respectively.

General

• Laboratory use

Isolation is made from early infection stages, small pieces of tissue being excised from the margins preferably of the youngest lesions. These are comminuted in small quantities of sterile water and streaked on plates of surface-dried King's medium B and incubated at 25-27°C for 2 days. The development of largely unmixed colonies, green fluorescent under UV light, is a good indication that the pathogen has been isolated. Single colonies are sub-cultured onto nutrient agar for storage and confirmation of the identity of the pathogen using LOPAT tests (Lelliott and Stead, 1987; Schaad et al., 2001). Presumptive diagnosis of the pathogen can be made if green fluorescent bacteria with the LOPAT Group III characteristics are obtained (being levan negative, oxidase positive, potato rot negative, arginine dihydrolase negative, hypersensitivity on tobacco positive). However, variable results for the LOPAT scheme were reported for the Pseudomonas strains designated as P. cichorii (Aysan et al., 2003; Cottyn et al., 2009). Culturally, P. cichorii most resembles the common plant saprophytes, P. fluorescens and P. putida. These are distinguished from P. cichorii because they are positive in the test for arginine dihydrolase and negative in the test for tobacco hypersensitivity. Further biochemical analysis can be performed according to Schaad et al. (2001) or by mean of galleries for biochemical characterization and identification. Pathogenicity tests could be performed on the host of isolation and preferably on one known host (e.g., lettuce or chrysanthemum). A bacterial suspension of no more than 10,000,000 CFU/ml is sprayed on plants until run off. Wounding is not necessary for leaf infection but high humidity levels (for example, enclosing plants in polyethylene bags) are essential until 24 h post inoculation. The plants are incubated at 26°C, being maintained for 3 days after inoculation in high humidity conditions, either by being enclosed with a polythene bag or, preferably, held in a mist cabinet. Plants may also be inoculated on stems using a needle laden with inoculum.

Two semi-selective media containing fungal and bacterial inhibitors have been developed for ecological studies on P. cichorii (Jones et al., 1990).

An ELISA procedure has been evaluated to detect P. cichorii in lettuce leaves (Ogiso et al., 1997). Polyclonal antibodies are commercially available for the identification of P. cichorii by immunofluorescence and ELISA. Any unusual report or diagnosis on plants or plant parts moving in trade should be confirmed by a pathogenicity test. Highly specific and sensitive molecular probes based on unique DNA sequences obtained by DNA subtraction methods can readily be developed to confirm the identity of the pathogen including strains obtained from symptomless plants. These methods are only used when a high level of confidence in identification is necessary to support eradication programmes or quarantine restrictions that may involve compensation.

Recently, a sensitive real‐time PCR assay was developed for the specific detection of P. cichorii using a Taqman Minor Groove Binding probe based on a 90‐bp amplicon from the pathogenicity gene cluster hrcRST. The method was shown to be capable of detecting 1 CFU/mL of irrigation water, which is well below the concentration needed for lettuce midrib rot infection in commercial lettuce greenhouses (Cottyn et al., 2011).

Inspect the plants or plant parts (e.g. cuttings) for the presence of leaf and/or stem lesions. Water-soaked lesions and bright-yellow halos at the margin of the lesions are indicative of P. cichorii. Symptoms on lettuce in the field are not always visible because the bacterium infects the inner leaves of mature heads but are evident when the heads are cut open (Grogan et al., 1977). Diseased specimens must be collected and analysed for the presence of the bacterium by isolation in culture and confirmation of identity.

Symptoms induced by P. cichorii can be confused with those caused by other bacterial pathogens that cause leaf spot or blight on vegetables and ornamentals. In tomato for instance, initial field symptoms caused by P. cichorii could be confused with the symptoms caused by P. syringae pv. tomato, P. corrugata as well as the leaf spot pathogen Xanthomonas euvesicatoria (Jones et al., 2016). In the laboratory, fluorescence of colonies on King's B medium restricts the investigation to fluorescent pseudomonads that can be identified by LOPAT tests (see Diagnosis).

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.

Control of disease caused by P. cichorii is difficult to achieve when conditions favour the pathogen. For greenhouse operations, the ability to regulate free water on plant surfaces offers some chance of success. Soil irrigation, rather than overhead watering, will reduce the likelihood of infection and disease development (Pauwelyn et al., 2011). Greenhouse ventilation is beneficial. Planting material should be from sources free of the pathogen. Plants with symptoms should be isolated if of high value, or discarded. The spread of the bacterium may be reduced by decontaminating tools and hands after handling diseased plants. High plant densities and over-fertilization, especially with nitrogen fertilizers, should be avoided. Inoculum in the field may be reduced by ploughing, removing and burning infected plant debris, removing weeds near growing areas. Different cultivar susceptibility has been observed in chrysanthemum (Strider and Jones, 1986), basil (Holcomb and Cox, 1998), endive, lettuce and chicory (Matta and Garibaldi, 1970). Chemical control using copper compounds or antibiotics has been demonstrated in trials, but success in commercial practice, especially in the field, is problematic. Spray costs and the variability of efficacy may mean that applications are often not cost effective.

Furthermore, resistance to streptomycin and copper-based chemicals was reported within the P. cichorii strains isolated from celery seedbeds in Florida, USA.

It has been shown that tomato seedlings inoculated with P. cichorii strain JBC1 and exposed to green or red light showed a significant reduction in disease incidence compared to the seedlings grown under white light or dark conditions. Inoculated seedlings grown under green or red light showed significant up-regulation in the defense-related genes, phenylalanine ammonia-lyase (PAL) and pathogenesis-related protein 1a (PR-1a) (Nagendran and Lee, 2015). Furthermore, Rajalingam and Lee (2018) showed that the expression of genes involved in the production of phytotoxic lipopeptides and siderophores were significantly decreased under green light in comparison to the dark conditions.

16/03/20 Reviewed by:

Ebrahim Osdaghi, Department of Plant Protection, College of Agriculture, Bajgah, Shiraz University, 71441-65186, Shiraz, Iran.