Seedborne Bacteria of Orange and Black Colour Carrots in Turkey

Article History Received: September 17, 2021 Revised: December 20, 2021 Accepted: December 27, 2021 Carrot (Daucus carota L.) is among the most economically important vegetable crops worldwide. Seedborne bacterial pathogens of carrot cause important damages to seed quality and yield of plants. In this study, seedborne bacteria were determined on some carrot seeds sown in Turkey. Seeds of different orange and black color varieties of carrot were collected from Eregli and Kasınhanı districts of Konya province, where the highest carrot production is reported. Subsamples of 10,000 seeds were soaked in 100 ml sterile saline (0.85% NaCl) with 0.02% Tween 20 overnight at 5 °C, YDCA, KB, MKM, MD5A and mTBM media were used for bacterial isolation and bacterial morphological characterization. Biochemical, physiological and molecular methods were used for the identification of the bacterial isolates. Pathogenicity tests of strains were performed on orange color carrots, and pathogenic strains induced a hypersensitive reaction in tobacco plants. The 60 pathogenic and saprophytic bacterial strains were obtained belong to Pseudomonaceae, Bacillaceae, and Xanthomonadaceae families. There were twentythree seed samples on 5 different orange carrot cultivars Maestro, Bolero, Sireco, Natuna and Romans, and 11 black carrot genotype of traditional cultivar ‘Eregli’. Two pathogenic bacteria were defined as Xanthomonas hortorum pv. carotae and Pseudomonas viridiflava at different percent infestation ratios (17.39-18.18%) and (9.09-13.04%) on orange and black carrot seed samples. To the best of our knowledge, it is the first report of P. viridiflava on carrot seeds in Turkey.


INTRODUCTION
Carrot (Daucus carota L.) is consumed as edible roots; usually, orange in color but red, white, purple, and yellow varieties also exist. Carrot is native to Europe and southwestern Asia and is a domesticated form of the wild carrot Daucus carota. Selective breeding has been practised for domestic carrot to obtain large, more delectable, and edible taproot with a less woody texture. Most carrot cultivars contain water (88%), sugar (7%), fibre (1%), ash (1%), protein (1%), and fat (0.2%) with almost no starch content. The fiber comprises mostly of cellulose, with smaller proportions of hemicellulose and lignin. Fructose, sucrose, glucose and xylose are free sugars found in carrots. Glutamic acid and other free amino acids are responsible for the taste while β-carotene mainly, and α-carotene and γcarotene to lesser extent, gives carrot the bright orange colour. In humans, α and β-carotenes are partly metabolized into vitamin A. Among carotenoid, βcarotene is the predominant while α-carotene and γcarotene are present in lesser amounts. Typically, there are between 6000 and 54,000µg of carotenoids in 100 grams of carrot root. There are abundant amounts of antioxidants and minerals in carrots having anti-aging, anti-inflammatory, antiproliferative, and anticarcinogenic properties.
Additionally, carrot consumption helps in maintenance of normal blood glucose and cholesterol levels, minimizes the risk of cardiovascular diseases, and protects against diabetes and Alzheimer's disease (Ahmad et al., 2019). According to the Food and Agriculture Organization of the United Nations (FAO), 44.76 million tonnes of carrots and turnips were produced worldwide in the calendar year 2019 (FAOSTAT, 2019). China led the chart with 21.48 million tonnes that accounted for 47.99% of the global output, followed by Uzbekistan, the USA and Russia, with almost 2.77, 2.26, and 1.56 million tonnes, respectively. Asia produced about 64.8% of the world's carrot followed by Europe (19%) and Americas (10%). Turkey is among the world's important carrot producers, with its annual production of 663.882 tonnes (TUIK, 2019). Bacteria, fungi and viruses can be transmitted through seeds in plants (Baker and Smith, 1966;Mundt and Hinkle, 1976). Thus one would expect natural selection to favor host plants that tightly control the kind and number of microbes that migrate into the developing seeds. Carrots are affected by several bacterial diseases; leaf blight (Xanthomonas hortorum pv. carotae), soft rot (Dickeya dadantii, Pectobacterium carotovorum subsp. carotovorum and Pectobacterium atrosepticum), crown gall (Rhizobium radiobacter), hairy root (R. rhizogenes), milky disease (Bacillus popilliae var. rhopaea) and scab (Streptomyces scabies) (Strandberg, 2000). Almeida et al. (2013) reported that Pseudomonas viridiflava was determined on carrot seeds, and this could explain how post-harvest damage might originate from contaminated seeds. The world's carrot seed industry is concerned directly with a seed-borne disease i.e., bacterial blight, caused by X. hortorum pv. carotae. Under optimum conditions, the quality of X. hortorum pv. carotae infested carrot seeds may reduce noticeably, which leads to a significant yield loss. Brown stem, blighted umbels, petiole lesions, necrotic leaf spots with irregular yellow halos, and gummy bacterial exudates on stems and umbels are typical symptoms of this disease. Use of X. hortorum pv. carotae free seed is important management practice as contaminated carrot seeds can be a potential primary source of inoculum. Therefore, inspection services, seed industry and commercial seed testing laboratories needed sensitive detection methods suitable for routine application (Meijerink and Van Breukelen, 1995). Despite being able to detect the seed-borne infection, development of seed contamination thresholds for specific regions of carrot production (Umesh et al., 1998), and the availability of seed treatments to eliminate seed-borne inoculum (Howard et al., 1994;Pscheidt and Ocamb, 2001), the carrot industry is still facing losses due to this disease (Kuan et al., 1985;Umesh et al., 1996). Studies regarding the prevalence of X. hortorum pv. carotae in carrot seeds in Turkey are very scarce and the pathogen was reported only once by Demir and Ustun (2001) in one carrot seed sample in Aegean Region. So it is important to study further the presence of this pathogen in carrot seeds in Turkey. The pectinolytic species Pseudomonas viridiflava is a multi-host and seed-borne pathogen causing severe damages to tomato (Alivizatos, 1986;Goumas et al., 1999), melon (Aysan et al., 2003) bean, birdsfoot, cabbage, cauliflower, kiwifruit, fennel, grape, lettuce, lupine, parsnip, passion fruit, pea, pepper, poinsettia, poppy, pumpkin, rape, soybean, and zinia, Amaranthus sp., Chrysanthemum sp., eggplant (Goumas et al., 1999), and Arabidopsis thaliana (Goss et al., 2005). Symptoms of P. viridiflava infection include yellowing of the plant and inner part of the stem, wilting, brown discoloration of vascular tissses and pith often developing soft rot. Particularly in Aegean islands, this pathogen is significant in the eastern Mediterranean region, representing 50% and 15%, respectively, of the Pseudomonas species causing stem necrosis (Aysan, 2001;Ustun and Saygili, 2001

Pathogenicity and hypersensitivity tests
Individual plastic pots containing 3 kg sterile soil were fertilized once with 9.5 g ammonium sulfate, 9.5 g diammonium phosphate, 9.5 g potassium sulfate, and 50 mL of a liquid humic acid (Kaçar and Katkat, 1999), and they were grown at 25 ± 5°C, 60-75% RH, and under 12.000-14.000 Lux from tungsten-filament lamps for a 16-h photoperiod. Five seeds were planted in each pot of each carrot variety ˈMaestroˈ at a depth of 8-10mm. Four week-old plants were sprayed with distilled water to create a fine humidity by a hand-held laboratory sprayer for bacterial development.
All suspect strains of X. h. pv. carotae were tested by atomizing the leaves to run off with a bacterial suspension containing 10 8 CFU ml -1 . Inoculated seedlings (five plants per strains) were incubated for 48 h to enhance infection. After this incubation, plants were set in a cool condition (at 20°C, 16 h) before placing the plants in greenhouse conditions at 25-30 °C with 90% relative humidity (Kuan, 1989;Asma et al., 2002). After the 15-day incubation period, treated seedlings were observed for pathogenicity based on typical leaves symptoms and intensity of stem necrosis (Taylor, 1970;Lelliott and Stead, 1987). Each strain was inoculated on two carrot plant leaves under same conditions. Finally, SDW and reference strains were sprayed on the control plants.
The inoculation of 26 carrot plants with tuberous roots was done by pricking the crown area of the root with a sterile needle after placing a drop of P. viridiflava suspension (10 8 CFU ml -1 ) to aid the bacterial entry into the tissues. SDW and reference strains were used to inoculate the control plants by the same method. Afterward, inoculated plants were transferred to a humid chamber for 72hrs followed by the transfer to the greenhouse. Daily examinations were conducted for 15 days to observe the symptom development. Carrot slices treated with SDW were used as controls.
Eight-week-old tobacco plants (Nicotiana tobaccum var. White Burley) were used to perform HR tests. The bacterial suspension (10 8 CFU ml -1 ) or water (control) was injected into the mesophyll cells using a 0.46-mmdiam (26-gauge) hypodermic syringe. Treated leaves were marked with numbers of the strains using a permanent pencil for evaluations. Three leaves of each tobacco plant were inoculated with each strain.

Determination of infestation ratios (%) of the pathogens on seed samples
The percent infestation for each carrot cultivar [PIP (%)] was calculated by commensurating the total infested seed samples (ΣISP) of relevant cultivar with the total collected seed (ΣSP) samples for each individual cultivar using following formula; The percent infestation of all cultivars in same group carrot (orange or black) [TI (%)] was determined by commensurating the total infested seed samples (ΣISR) of relevant all cultivars in the same group carrot (orange or black) with the total collected seed samples (ΣSR) for that region using the following formula; (%) = Ƹ Ƹ × 100

RESULTS
Total 60 bacterial isolates were obtained from twenty three seed lots on 5 different orange carrot cultivars, Maestro, Bolero, Sireco, Natuna and Romans, and 11 black carrot genotype of traditional cultivar 'Ereğli' in Konya province. Identification of bacteria was done by biochemical, morphological, physiological (Table 2) and molecular methods. According to our findings, mainly two pathogenic bacteria were identified as X. h. pv. carotae (16 strains) and P. viridiflava (13 strains) on orange and black carrot seeds at different percent infestation ratios (Table 3).

Biochemical and physiological characteristics
Total twenty-nine X. hortorum pv. carotae and P. viridiflava strains isolated from orange and black carrot seeds were identified by biochemical and physiological tests ( Table 2). Sixteen X. hortorum pv. carotae strains gave negative results from fluorescent pigment production on KB medium, facultative growth and pectolytic activity test. However, they were positive for starch and gelatin hydrolisis, and H2S production from cysteine. The strains did not produce acid from adonitol, arabinose and sorbitol. Almost all strains gave identical results in these tests and the biochemical profiling to the X. hortorum pv. carotae reference strain. Obtaining data, only two (Xhcbc14 and Xhcoc3) out of twenty-one X. h. pv. carotae strains gave different results for gelatin hydrolysis test compare to reference strains ( Table 2). All strains were positive for catalase test and negative for oxidase and arginine dihydrolase activity tests. The strains were able to produce acid from sorbitol but not from sucrose. The strains were positive for pectolytic activity based on the ability to cause soft rot on potato slices. Results of these tests along with biochemical profiling of the test isolates were identical to the reference strain of P. viridiflava and were differentiated from negative references (Table 2).
Pathogenicity and HR reaction of X. hortorum pv. carotae and P. viridiflava strains At first, the symptoms of X. hortorum pv. carotae on inoculated leaves appeared as small irregular yellowish water-soaked lesions with a tiny light brown spot in the center, which, in case of leaf spots enlarged at a later stage and became necrotic surrounded by a yellow halo. Eleven out of sixteen X. hortorum pv. carotae strains were pathogenic on leaves of cv. Maestro. Re-isolation of bacteria from inoculated plants confirmed the same as the inoculated ones. Areas observed around the points of inoculation with P. viridiflava were light to dark brown. The roots' degradation was seen progressing from crown to the root tip when longitudinal sections were examined. Nine P. viridiflava strains out of thirteen were pathogenic on the roots on cv. Maestro. Re-isolations made from the artificially infected plants yielded pure cultures.
In HR tests, all bacterial strains that caused necrosis on tobacco leaves and brown, collapsed areas of tissues were observed at the injection sites after 48 h of incubation at 28 ℃ and 80% RH.

PCR Assays
Characterization of X. hortorum pv. carotae and P. viridiflava strains by PCR showed that all X. hortorum pv. carotae strains amplified with the primers 3SF and 3SR and almost all P. viridiflava strains (except str. PvBc6) reacted with the primers PVF/ PVR. X. hortorum pv. carotae strains gave a product size of 350 bp with the specific primers, 3SF and 3SR. An 860 bp PCR amplified fragment of P. viridiflava strains using PVF1/PVR1 primers designed for 16S rRNA sequence.

DISCUSSION
Carrot is among the top-ten economically important crops worldwide. According to agricultural statics, Turkey has an important place in the world carrot production (TUIK, 2019). Seventy percent of Turkey's carrots are produced in Konya province and are exported to the Balkans countries and Middle East (Livaneli, 2011). Bacterial blight of carrot, caused by seedborne X. hortorum pv. carotae bacterial pathogen (Kendrick, 1934), concerns carrot growers in many regions of the world (Gilbertson, 2002;Cubeta and Kuan, 1986;Watson, 1948;Saad and Wade, 1972;Nishiyama et al., 1979;Meng et al., 2004). The pathogen can infect the seed internally or contaminate the seed surface, affecting the yield (Ark and Gardner, 1944;Umesh et al., 1998). Reduction of seed germination due to the infection by X. hortorum pv. carotae can result in significant losses to the farmers (Crowe and Bafus, 2004). Unfortunately, there is insufficient knowledge about the epidemiology of X. hortorum pv. carotae about carrot seeds. It is thought that the surface infestations probably take place during seed development in umbel and harvesting operations. True infection of inner seed tissues would start during early seed development, perhaps during the living connection of seed with mother plant or via pollen or nectar (Bashan and Okon, 1986). Since the Central Anatolia region has hot and dry conditions, bacterial diseases are seen at very low rates, and therefore, the symptoms of the disease are often not noticed in field conditions which are mistaken for the absence of the pathogen. In this regard, it should be emphasized that the disease can reach serious dimensions, especially in high irrigation conditions, humid and cool regions, and regions with different climatic conditions. Melon and tomato crops foliage can be reduced up to 50% as a result of infection by P. viridiflava that causes extensive tissue necrosis (Goumans and Chatzaki, 1998). The opportunistic behavior of P. viridiflava allows it to survive on surfaces of its host or other weeds that can act as an inoculum source. P. viridiflava was responsible for severe economic losses to refrigerated carrot export from New Zealand by causing soft rot (Godfrey and Marshall, 2002). In Brazil, symptoms of P. viridiflava during post-harvest storage on potatoes were described by Macagnan et al. (2007) and observed the infection symptoms on chayote, sweet potato, yam, and pumpkin when inoculated the pathogen artificially. Association of P. viridiflava with carrot seeds may explain that postharvest losses can originate from contaminated seeds (Almeida et al., 2013). Demir and Ustun (2001) tested a total of two thousandfour hundred-sixty-nine plant propagative units for the presence of some plant pathogenic bacteria. They detected Pseudomonas viridiflava in 3 samples (cauliflower and cabbage seeds), X. hortorum pv. carotae in one carrot seed sample in Aegean Region. Kurt et al. (2004) obtained Erwinia spp. and fluorescent and nonfluorescent plant pathogenic Pseudomonas spp. strains from diseased carrot roots, in the Eastern Mediterranean region. In present study, to the best of our knowledge, this is the first report of P. viridiflava associated with carrot seeds in Turkey. As like X. h. pv. carotae, a high level of infestation was determined on orange and black carrot seeds by P. viridiflava as 9.09% and 13.04%, respectively. Lack of such studies increases the significance of the present work in Central Anatolia. The production of commercial carrot crops depends greatly on planting seeds with zero or low contamination of X. hortorum pv. carotae. In this study, high infestation ratios of X. hortorum pv. carotae on orange and black carrot seed samples were determined, between 17.39% and 18.18% respectively (Table3). In that region, most cultivated orange carrot cultivar is Maestro, and according to findings, this cultivar was infested with both X. hortorum pv. carotae and P. viridiflava. In order to control these diseases, necessary measures should be considered timely. Twenty three orange and black carrot seed samples were infested with saprophytic bacteria, Pseudomonads and Bacillus spp. They did not show any antagonistic effect on pathogenic bacteria under in vitro conditions but they can cause decay on seeds at non-suitable storage conditions because of their high infestation (%) in the seeds (18.18% -86.95%) respectively (Table 3). The standard procedure for detection currently in use includes seed-wash dilution plating assay, together with pathogenicity tests of representative X. hortorum pv. carotae colonies. Although effective in general, this procedure is laborious and can take as long as one month to complete (Meng et al., 2004). In the present study, King's B, Nutrient Agar, MD5A, MKM, mTBM, and YDCA media were used for isolation and characterization of bacterial colony morphology. The strains were identified by biochemical, physiological pathological and HR tests for all X. hortorum pv. carotae, P. viridiflava and saprophytic bacteria. Thus, development of more rapid PCR-based methods for detection and identification of seed contaminated by the pathogens should improve the capacity to detect and manage carrot bacterial diseases. PCR with the 3S primer pair could greatly reduce the time and facilities required for routine detection and identification of X. hortorum pv. carotae and could be used in ecological and epidemiological studies (Meng et al., 2004). In this study, the 3S primer pair were highly specific, and the about 350-bp target fragment amplified from all strains. Likewise PVF/PVR primers set confirmed P. viridiflava strains. The observations of present study suggested that PCR experiments were same to those reported by Goss et al. (2005). The PCR-based assays have the potential to improve routine detection of important carrot pathogens. Epiphytic growth of X. hortorum pv. carotae is promoted under semi-arid areas where carrot seeds are produced (du Toit et al., 2005). The bacterial association with the seeds acts as an inoculum to cause bacterial blight when carrots are grown in humid and warm areas. Carrot seed produced in Turkey harbors X. hortorum pv. carotae so there is a dire need to identify the primary inoculum source to avoid future infections when grown under semi-arid regions of Turkey. Identifying these sources of infection and the primary periods of infection through the biennial season will assist in the development of more efficacious, regional integrated pest management programs for carrot seed crops. Black carrots are a source for natural food coloring from Turkey and other regions of the Middle East and Asia. In Turkey, black colour carrot is produced in only Ereğli district of Konya Province and the growers use the genotypes of cv. Ereğli as seed. This situation contains two important problems; first, genetic expansions and susceptibilities to plant diseases as the same seeds are being used for many years. Second, transmission of fungal, bacterial and viral pathogens, both plant and human pathogens, from year to year and field to field. Further studies will be conducted to determine pathogens related to plant and human health, carrot yield, and orange carrots. Plantation of healthy seed or treated seed can be an important strategy to manage bacterial blight (Umesh et al., 1998;Gilbertson, 2002;Meng et al., 2004). Other management recommendations include 2 to 3 year crop rotations, applications of copper bactericides, avoiding overhead irrigation that promotes dispersal of the bacterium and creates favorable conditions for infection and incorporating residues into the soil promptly after harvest (Parks and Crowe, 1999;Gilbertson, 2002;Weber et al., 2004). Seed can be disinfected by hot water treatment (52 °C for up to 25 min) (Ark and Gardner, 1944;Gilbertson, 2002;Pscheidt and Ocamb, 2001). Limited resistance to bacterial leaf blight is available in some commercial cultivars. There is no study to determine susceptible or resistant carrot cultivars to fungal, bacterial or viral diseases in Turkey. This will become very important for future.
In subsequent experiments, authors plan to focus on the use of resistant cultivars for carrot bacterial diseases. Cultivar resistance is the most desirable for its costeffectiveness and long-term stability. The development of resistant cultivars requires advanced planning in breeding programs to incorporate and maintain a diverse range of resistance genes in parental lines, but little is known about carrot resistance to bacterial diseases. Therefore, further studies should be conducted primarily between carrot varieties and X. hortorum pv. carotae and P. viridiflava interactions.

CONCLUSION
Bacterial pathogens of carrot cause important damages on plant yield and seed quality. This study was conducted for detection and identification of seed borne bacterial pathogens on orange and black carrot seeds sown in Ereğli and Kaşınhanı districts of Konya province where the highest carrot production is obtained. Based on morphological, physiological, biochemical, pathological and molecular tests, the pathogenic agents were identified as Xanthomonas hortorum pv. carotae and Pseudomonas viridiflava with high percent infestation ratios. This to our knowledge is the first report of the occurrence of P. viridiflava on carrot seeds in Turkey. The information will be useful about using carrot seeds and required precautions to growers and authorities to get more healthy products and seeds.