In 1999, New Zealand carrots (Daucus carota) exported to the Middle East incurred substantial damage due to bacterial soft rot that resulted in major financial loss to farmers. A modified carrot tissue bioassay was developed under standard conditions to provide an economical and rapid means of monitoring export carrots for bacteria able to cause severe bacterial soft rot. Using this bioassay, two bacterial isolates causing severe degradation were detected and subsequently identified as Pseudomonas viridiflava (designated NZCX09) and P. marginalis (NZCX27), using the Biolog system and 16S rRNA phylogenetic analysis. Investigation of disease epidemiology of NZCX09 and NZCX27 at low temperatures showed that tissue degradation occurred at temperatures approaching 0°C. These findings emphasize the importance of postharvest sanitization, and the efficacy of refrigeration methods used in controlling soft rots in carrots stored over time.
The carrot (Daucus carota) root is an economically important horticultural crop that is harvested from the soil while in full metabolic activity. The aim of postharvest storage is to maintain carrots in the same condition as at the time of harvest. Two methods commonly used for carrot storage are field storage and refrigerated storage. Field storage involves leaving carrots in the ground over winter with the tops covered by straw as protection from frost. However, while field storage is cheaper than refrigerated storage, this practice can lead to significant loss of quality, especially if there are severe frosts or if wet spring weather delays harvesting (Geeson et al., 1988). Refrigerated storage is preferred, especially in temperate countries, and carrots maintained in refrigerated storage are generally considered to be of better quality than the field-stored crop (Bedford, 1982). For many years it has been thought that long-term refrigerated storage requires two essential environmental conditions: maintenance of temperature at 1°C, and high relative humidity at 98% (Hasselbring, 1927; Lentz, 1966; Van den Berg & Lentz, 1966; Phan et al., 1973). These conditions have been reported to enable high-quality storage of undamaged roots for up to 7 months (Geeson et al., 1983; Geeson, 1984). By preventing moisture loss from the roots, high-humidity storage not only preserves quality, but also reduces susceptibility to invasion by pathogens (Snowdon, 1991).
Although refrigerated storage has proved successful in the maintenance of carrot freshness, there are documented cases of problems associated with long-term storage (Geeson et al., 1983). One of the major problems has been the high wastage and restricted storage life attributed to severe microbiological spoilage (Derbyshire & Crisp, 1978). Carrot root spoilage due to microbial disease is well documented, and can be caused by a number of soilborne microorganisms including many species of fungi, yeasts and bacteria (Snowdon, 1991). Spoilage of carrots has been attributed to predisposing factors including: (i) the presence of organic debris or soil; (ii) the degree of wounding from mechanical harvesting and prestorage washing; or (iii) the length of the cold-storage period (Goodliffe & Heale, 1977).
Bacterial soft rots of carrots and parsnips have been reported worldwide, including the USA (Burkholder & Smith, 1949; Hunter & Cigna, 1981); the former USSR (Matveeva, 1982); New Zealand (Dye, 1953); Italy (Tamietti & Matta, 1981); and Romania (Tasca et al., 1975). Bacterial soft rot lesions may occur anywhere on individual roots, although infection often occurs at the crown or the root tip and then progresses rapidly through the core region (Hunter & Cigna, 1981; Towner & Beraha, 1976). Infected flesh is often brownish in colour and may be extremely soft and ‘slimy’ to the touch. A putrid odour is often associated with soft rot, and is due to secondary infection(s) by bacteria. Furthermore, soft rots are frequently colonized by secondary fungal infection(s) which mask the primary cause.
Pseudomonas marginalis is an opportunistic phytopathogen responsible for a large proportion of postharvest rots in cold storage (Lund, 1983), and at wholesale and retail markets (Laio & Wells, 1987). Apart from its disease-causing ability, P. marginalis is physiologically indistinguishable from various biovars of P. fluorescens (Lelliott et al., 1966). The ability of P. marginalis, P. viridiflava and other soft-rotting bacteria to cause maceration of plant tissue is mainly due to their production of an array of enzymes required for degradation of pectic components in plant cell walls (Collmer & Keen, 1986; Liao et al., 1988).
Because carrots are marketed as a fresh vegetable and used in the manufacture of a wide range of processed foods, the demand is high. In many countries this demand cannot be met by home-grown crops, and carrots are imported. New Zealand exports carrots, and this market is quickly becoming lucrative. However, because the distances between New Zealand and importing countries are large, carrots remain in refrigerated storage for considerable periods. Export of carrots in conventional refrigerated containers requires maintenance of a carriage temperature of 1°C (Snowdon, 1991). Not all refrigerated ships and containers are suited to this task, and fluctuating temperatures can lead to the establishment of disease during transport.
This study was initiated because New Zealand exported carrots arriving in the Middle East exhibited bacterial soft rot symptoms of surface tissue degradation, with a viscous, ‘slimy’ residue (Fig. 1) and a sweet, pungent smell. Importers have rejected carrots arriving in this condition, and therefore bacterial soft rots have become a financial concern in New Zealand. This paper (i) identifies two bacterial isolates, P. viridiflava NZCX09 and P. marginalis NZCX27, causing soft rot on New Zealand exported carrots; (ii) describes a modified bioassay used to rapidly identify bacterial soft rot; and (iii) reports the efficacy of refrigeration temperatures in controlling the soft rot severity of NZCX09 and NZCX27.
Materials and methods
Bacterial strains and culture conditions
Bacterial isolates used in this study are listed in Table 1. All strains used in this study were maintained at –80°C in Luria Bertani medium (LB) (Difco Laboratories, Detroit, MI, USA) containing 20% (v/v) glycerol. Strains were transferred onto nutrient agar (NA) (Remel, Lenexa, KS, USA), incubated at 28°C for 16 h and then maintained at 4°C for short-term use.
Table 1. Bacterial strains and species used in this study
(a) Bacterial strains used in this study; (b) 22 validly described Pseudomonas (sensu stricto) (Moore et al., 1996) used in 16S rRNA gene phylogenetic analysis; (c) Acinetobacter calcoaceticus included for single sequence (forced) outgroup rooting of the tree.
ATCC, American Type Culture Collection, Rockville, MD, USA.
DSM, Deutsche Sammlung von Mikro-organismen, Gottingen, Germany.
LMG, Laboratorium voor Microbiologie en Genetica, Rijksuniversiteit, Gent, Belgium.
IAM, Institute of Applied Microbiology, Tokyo, Japan.
NCPPB National Collection of Plant Pathogenic Bacteria, York, UK.
Carrots exhibiting severe bacterial soft rot disease symptoms were obtained during August 1999 either from packaged carrots in cold store awaiting export shipment from New Zealand, or from carrots returned from the Middle East exhibiting severe bacterial soft rot. Carrots were swabbed using the sterile BBL CultureSwab Plus collection and transport system for aerobes and anaerobes (Becton Dickinson, Franklin Lakes, NJ, USA). Swabs were placed into sterile 0.84% saline solution (2 mL) and vortexed (20 s). At this stage, 1 mL saline solution containing bacteria was stored as above for whole-carrot bioassays. The remaining 1 mL saline solution was serially diluted using 0.84% saline solution (to create a dilution series 10, 10−3, 10−6 and 10−9). An aliquot (100 µL) of each dilution was spread-plated until dry on NA. After 24 h incubation at 28°C, the resulting individual colonies were purified by passage in duplicate onto fresh NA and Kings B (KB) (King et al., 1954) media to select for potential pseudomonads. Purified bacterial isolates were stored as described above.
Whole-carrot bioassays to simulate export conditions
Whole-carrot bioassays were used initially to fulfil Koch’s postulates. Either (i) saline solutions containing a suspension of total naturally occurring bacterial populations (described above); or (ii) purified bacterial isolates (cultured for 16 h at 28°C) in LB medium to a concentration of ≈1 × 109 colony-forming units (CFU) mL−1, were inoculated onto healthy carrots using a sterile cotton swab (Becton Dickinson). Ten healthy carrots were inoculated and placed randomly among other healthy carrots in plastic-lined export cardboard cartons (on average each 20 kg carton contained ≈100 medium-grade carrots). Cartons of uninoculated carrots were set up as negative controls. Cartons were incubated at the required temperatures and analysed after 7 days by visual assessment of tissue decay of both inoculated whole carrots, and carrots in close proximity to inoculated carrots.
Sliced carrot bioassay to determine virulence of bacterial isolates
The pathogenic potential of bacterial cultures from soft rot lesions was determined using a modification of the bioassay method previously described by Gandy (1968) for determining pathogenic pseudomonads of the mushroom Agaricus bisporus. Healthy carrots were obtained from the packaging line and submerged in sodium hypochlorite providing 1% available chlorine for a period not exceeding 1 min, to eliminate surface microorganisms. A slice of carrot (≈5 mm) was obtained using a sterile scalpel blade and placed in a sterile Petri dish containing a 50 mm paper filter dampened with sterile double-distilled water (800 µL). A 50 µL aliquot of either (i) saline solutions containing total bacterial suspensions isolated from diseased carrots; or (ii) purified bacterial isolates (cultured for 16 h at 28°C) in LB medium to a concentration of ≈1 × 109 CFU mL−1; or (iii) uninoculated control, was placed onto a carrot slice within a Petri dish and incubated (sealed and undisrupted) at the required temperatures. Bioassays were analysed at 24, 48 and 72 h intervals by visual assessment of tissue decay in each carrot slice.
Effect of temperature on growth of bacteria in vitro
McCartney tubes containing 10 mL LB broth were inoculated with 1000 CFU (±68, determined by serial dilution-plate assays) from a 16 h culture of the respective bacterium, giving a final inoculation concentration of ≈1 × 102 CFU mL−1. Four inoculated McCartney tubes were placed at each of the following temperatures: –3, 0, 2, 4, 22, 28, 40, 45 and 50°C. Cultures were grown statically as shaking was not possible at all temperatures. Following 24 h incubation, cultures were serially diluted and spread-plated onto LB agar, and colonies were counted following incubation (16 h, 28°C) to determine CFU mL−1. The mean of four CFU counts was obtained for each bacterium at each respective temperature.
The Biolog system
The Biolog GN2 MicroPlate identification system for gram-negative bacteria was used according to the manufacturer’s instructions (Biolog Inc., Hayward, CA, USA).
Genomic DNA isolation, PCR and DNA sequencing
DNA was isolated from pure cultures of bacteria using the Wizard Genomic DNA Isolation Kit (Promega, Madison, WI, USA). PCR amplifications were carried out in a Perkin Elmer 9700 thermocycler (Perkin Elmer, Auckland, New Zealand). Unless stated otherwise, the standard PCR reaction mixture (25 µL total) consisted of 1× buffer (10 mm Tris-HCl pH 9.0, 50 mm KCl, 2.5 mm MgCl2, 0.01% gelatin and 0.1% Triton X-100), deoxyribonucleotide triphosphates (dATP, dCTP, dGTP, dTTP) at a final concentration of 200 µm, 0.6 U Taq DNA polymerase (Roche Molecular Biochemicals, Mt Wellington, Auckland, New Zealand), oligonucleotide primers (Table 2) at a final concentration of 2 µm, and 100 ng template DNA. PCR consisted of 30 cycles of 1 min 94°C, 1 min 55°C, and 1 min 72°C. Prior to cycling, samples were heated at 94°C for 5 min and, as part of the terminal cycle, the extension step was increased to 5 min, 72°C. Contaminating primers and dNTPs were removed from PCR products using the High Pure PCR Product Purification Kit (Roche Molecular Biochemicals). PCR amplicons were directly sequenced with respective oligonucleotide primers (Table 2) using the Big Dye Terminator Kit and ABI Prism 3TIXLCPE (PE Biosystems, Foster City, CA, USA). All 16S rRNA genes sequenced in this study were confirmed by determining contiguous overlapping sequences of PCR-DNA produced. The nearly complete 16S rRNA gene sequences determined in this study have been deposited with the GenBank Database under accession numbers listed in Table 1.
Table 2. Oligonucleotide PCR primers used in this study
Whole-carrot bioassays of naturally occurring populations of bacteria from exported diseased carrots
All healthy whole carrots that had been swabbed with saline solution containing total bacterial suspensions isolated from diseased carrots exhibited disease symptoms (surface tissue degradation with a viscous, slimy residue and a sweet, pungent smell) after 7 days, within 20 kg export cartons. Carrots in contact with inoculated carrots also exhibited disease symptoms. Disease symptoms from cartons of carrots incubated at 0–1°C (refrigerated shipping containers) were comparable to those observed with diseased exported carrots from which the bacterial inoculum originated (Fig. 1). Cartons left at higher temperatures (5, 18 and 25°C) exhibited relatively more severe disease symptoms as incubation temperatures increased.
Isolation and identification of bacterial soft rot isolates
Thirty-two bacterial colonies were isolated from total bacterial suspensions from carrot soft rot lesions. These were purified, and REP-PCR was performed to eliminate duplicate strains. Eight distinct REP-PCR profile groupings emerged (data not shown), and one isolate from each of these groupings was subjected to the sliced carrot tissue bioassay to determine individual pathogenic potential. The bioassay identified two bacterial isolates that caused aggressive carrot tissue degradation at ambient temperature (Fig. 2). These two isolates were designated NZCX09 and NZCX27. These isolates were subsequently shown to cause the same aggressive carrot tissue degradation in whole-carrot bioassays under export packaging conditions.
Bacterial identification of NZCX09 and NZCX27
The Biolog system identified NZCX09 as P. viridiflava and NZCX027 as P. marginalis. The inferred phylogenetic relationships derived from 16S rRNA gene nucleotide sequences, and the neighbour-joining analysis of pairwise comparisons shows P. marginalis NZCX27 to cluster (89%) with the typed P. marginalis pv. marginalis LMG 2210T, and P. viridiflava NZCX09 to cluster tightly (100%) with P. viridiflava LMG 2352T (Fig. 3).
Effect of temperature on sliced carrot tissue degradation
Carrot tissue bioassays carried out at 10, 3 and 1°C showed that, although tissue degradation was reduced in comparison to replicate ambient temperature bioassays, at these lower temperatures tissue degradation still occurred and resulted in a ‘slimy-smooth to touch’ texture on the carrot surface compared with negative controls (Fig. 4).
Effect of temperature on NZCX09 and NZCX27 growth in LB broth
Low-temperature tolerance of NZCX09 and NZCX27 was further supported by the observation that after 24 h incubation in LB growth, the CFU mL−1 of both NZCX09 and NZCX27 had increased, even at temperatures approaching 0°C (Table 3).
Table 3. Concentration (CFU mL−1) obtained by inoculating 1 × 102 CFU mL−1 of Pseudomonas viridiflava (NZCX09) or P. marginalis (NZCX27) in 10 mL LB broth after incubation for 24 h at different temperatures
Bacteria isolated from carrots exhibiting soft rot in this study yielded two isolates able to cause severe tissue degradation in carrot tissue bioassay (NZCX09 and NZCX27). NZCX09 and NZCX27 were initially identified as P. viridiflava and P. marginalis, respectively, using the Biolog system; a previous report suggests this system does accurately recognize these two species (Lacroix et al., 1995). Confirmation of NZCX09 and NZCX27 speciation was further sought by analysis of the 16S rRNA gene, as this is also considered an effective method for defining prokaryotic genotypic relatedness and resolving taxonomic identities (Fox et al., 1980; Head et al., 1998; Moore et al., 1996). The 16S rRNA nucleotide sequences of NZCX09 and NZCX27 were compared with 22 pseudomonads validly described in a comprehensive phylogenetic study (Moore et al., 1996). The identification of NZCX09 and NZCX27 is in agreement with previous studies identifying P. viridiflava and P. marginalis as the causal agents of bacterial soft rots of both carrots and parsnips (Hunter & Cigna, 1981).
Koch’s postulates were fulfilled using the whole-carrot bioassays under export conditions to establish that NZCX09 and NZCX27 were able to cause disease (i) within naturally occurring bacterial populations isolated from soft rot lesions; and (ii) as purified cultures. Following the identification of NZCX09 and NZCX27, export carrot shipments in cold store were routinely monitored from a major New Zealand processing plant for soft-rot disease symptoms. From these soft rot lesions, P. viridiflava and P. marginalis strains were isolated with identical characteristics to NZCX09 and NZCX27 (16S rRNA sequence, Biolog, REP-PCR and temperature tolerance bioassays). This suggested that NZCX09 and NZCX27 were the causal agents of bacterial soft rots of carrots from this New Zealand processing plant. Furthermore, NXCX09 and NZCX27 were shown to be present throughout the processing plant, providing potential inoculation points prior to packaging of carrots before export (unpublished results).
Soft rot bacteria are reported to survive on plant debris in the soil (Matveeva, 1982) and to infect growing crops of carrots (Towner & Beraha, 1976) which, in addition to causing rotting in field-stored carrots (Tamietti & Matta, 1981), can be the source of postharvest contamination of carrots. A build-up of bacteria in washing tanks has been identified as a source of postharvest inoculation (Segall & Dow, 1973; J.W.M. and S.A.C.G., unpublished results). The outer cuticle of the carrot is physically removed within washing tanks to improve aesthetic quality and market value, and it is likely that this treatment would facilitate infection by soft rot bacteria.
Because a method for identifying bacterial isolates pathogenic to carrots is essential in investigating bacterial soft rot, this study developed a method for rapidly identifying highly pathogenic bacterial isolates under standardized conditions, with a focus on economy of materials and time. As this method requires only standard sterile laboratory equipment and appropriate means to culture bacterial isolates, it is economical and allows numerous isolates to be screened in replicate. The carrot-slice bioassay accurately discriminated between pathogenic and nonpathogenic bacterial isolates, and also identified different degrees of tissue degradation. The contained nature of the bioassay allows it to be easily subjected to various temperatures. Bioassays at low temperatures showed that NZCX09 and NZCX27 caused tissue degradation at refrigeration temperatures, and this has serious implications for postharvest sanitization. These results indicate that refrigeration temperatures alone are not enough to prevent the growth and formation of soft rot by pathogenic bacteria such as NZCX09 and NZCX27. It is therefore essential to focus on eliminating such bacteria from the carrots prior to packaging.
NZCX09 and NZCX27 were isolated from carrots grown in the South Island of New Zealand where the soil is consistently exposed to lower temperatures. It is likely that NZCX09 and NZCX27 have been exposed to a strong evolutionary selection pressure, so cold tolerance would be a metabolic advantage and would favour survival. While most microorganisms reduce metabolic activity under refrigeration temperatures to a rate that eliminates spoilage, having an environmental adaptation to cold tolerance may explain the ability of NZCX09 and NZCX27 to cause disease at low temperatures. Tissue degradation was enhanced at temperatures above 1°C in this study, so concern must focus on temperature fluctuations during shipment. Temperature fluctuations would favour growth of secondary microbial pathogens entering the primary lesions created by P. marginalis and P. viridiflava isolates, and would worsen overall disease conditions of carrots in transport.
In conclusion, the modified bioassay presented in this study enables rapid screening of numerous carrots for bacteria causing soft rot. Such a method will enable routine monitoring of the efficacy of postharvest sanitization in carrot-processing plants; identify specific sites of contamination; and ultimately prevent the severe bacterial soft rot during extended export shipments. Bioassay detection of P. viridiflava NZCX09 and P. marginalis NZCX27 causing aggressive tissue degradation, even under refrigeration temperatures, highlights the need for effective postharvest sanitization for the elimination of these organisms before export packaging, as cold-store conditions will not prevent the establishment of disease.