Evaluating Hawaii-grown Papaya for Resistance to Internal Yellowing Disease Caused by Enterobacter cloacae

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  • 1 U.S. Department of Agriculture, Agricultural Research Service, U.S. Pacific Basin Agricultural Research Center (PBARC), P.O. Box 4459, Hilo, HI 96720
  • | 2 College of Tropical Agriculture and Human Resources, University of Hawaii-Manoa, 3050 Maile Way, Honolulu, HI 96822

Papaya (Carica papaya L.) cultivars and breeding lines were evaluated for resistance to Enterobacter cloacae (Jordan) Hormaeche & Edwards, the bacterial causal agent of internal yellowing disease (IY), using a range of concentrations of the bacterium. Linear regression analysis was performed and IY incidence was positively correlated with increasing inoculum concentrations for susceptible cultivars Kapoho Solo and Laie Gold but not for resistant cultivars or lines. It was determined that the inoculum concentration of 9 to 10 Log10 colony-forming units per milliliter (cfu/mL) was able to reliably differentiate resistant and susceptible papaya germplasm. Red-fleshed cultivars SunUp and Sunrise were the most resistant papaya groups evaluated at this dose concentration. Yellow-fleshed cultivars, Kapoho Solo and Laie Gold, were susceptible to E. cloacae. ‘Rainbow’, an F1 hybrid between ‘SunUp’ and ‘Kapoho Solo’ that is yellow-fleshed, was moderately resistant to E. cloacae, exhibiting limited symptoms of the disease. Yellow-fleshed I-Rb F5/F6, an advanced inbred line derived from ‘Rainbow’, is resistant and offers the potential of improving resistance of yellow-fleshed commercial cultivars. A colorimeter was used to objectively measure internal flesh color and distinguish between infected and non-infected tissue in red- and yellow-fleshed papayas using L*C*H* color space analysis. Symptomatic tissue (72.4 and 79.0°) had higher hue angle means than non-symptomatic tissue (62.8 and 75.0°) for all cultivars or lines in red- and yellow-fleshed papayas, respectively. Yellow (“Y”) hue color also distinguished infected tissue from non-infected tissue. Symptomatic tissue that had Y hue color resulted in 79 to 81° hue angle means among red- or yellow-fleshed papayas. Our results demonstrated the usefulness of colorimetry to help detect infected papaya tissue. In surveys of naturally infected papaya, high populations (8.57 × 107 cfu/g) of E. cloacae were recovered in infected fruit of ‘Kapoho Solo’ and represent a food safety concern for fresh and processed papaya. In isolations from inoculated fruits, we observed decreases of ≈1 to 2 Log10 cfu/g in final bacterial populations when high-dose range inoculum concentrations (9 to 12 Log10 cfu/mL) were used. This dose range may represent a saturation range for E. cloacae inoculation.

Abstract

Papaya (Carica papaya L.) cultivars and breeding lines were evaluated for resistance to Enterobacter cloacae (Jordan) Hormaeche & Edwards, the bacterial causal agent of internal yellowing disease (IY), using a range of concentrations of the bacterium. Linear regression analysis was performed and IY incidence was positively correlated with increasing inoculum concentrations for susceptible cultivars Kapoho Solo and Laie Gold but not for resistant cultivars or lines. It was determined that the inoculum concentration of 9 to 10 Log10 colony-forming units per milliliter (cfu/mL) was able to reliably differentiate resistant and susceptible papaya germplasm. Red-fleshed cultivars SunUp and Sunrise were the most resistant papaya groups evaluated at this dose concentration. Yellow-fleshed cultivars, Kapoho Solo and Laie Gold, were susceptible to E. cloacae. ‘Rainbow’, an F1 hybrid between ‘SunUp’ and ‘Kapoho Solo’ that is yellow-fleshed, was moderately resistant to E. cloacae, exhibiting limited symptoms of the disease. Yellow-fleshed I-Rb F5/F6, an advanced inbred line derived from ‘Rainbow’, is resistant and offers the potential of improving resistance of yellow-fleshed commercial cultivars. A colorimeter was used to objectively measure internal flesh color and distinguish between infected and non-infected tissue in red- and yellow-fleshed papayas using L*C*H* color space analysis. Symptomatic tissue (72.4 and 79.0°) had higher hue angle means than non-symptomatic tissue (62.8 and 75.0°) for all cultivars or lines in red- and yellow-fleshed papayas, respectively. Yellow (“Y”) hue color also distinguished infected tissue from non-infected tissue. Symptomatic tissue that had Y hue color resulted in 79 to 81° hue angle means among red- or yellow-fleshed papayas. Our results demonstrated the usefulness of colorimetry to help detect infected papaya tissue. In surveys of naturally infected papaya, high populations (8.57 × 107 cfu/g) of E. cloacae were recovered in infected fruit of ‘Kapoho Solo’ and represent a food safety concern for fresh and processed papaya. In isolations from inoculated fruits, we observed decreases of ≈1 to 2 Log10 cfu/g in final bacterial populations when high-dose range inoculum concentrations (9 to 12 Log10 cfu/mL) were used. This dose range may represent a saturation range for E. cloacae inoculation.

Although the popularity of convenient, packaged, cut fruit is increasing, there are also concerns over foodborne diseases and spoilage. Fresh-cut and frozen papaya preparations have potential as value-added products of high quality for the papaya industry of Hawaii. However, high coliform bacterial counts have led to unacceptable batches of frozen cube preparations of ‘Kapoho Solo’ papaya fruit (Nishijima, unpublished data). The source of the high counts was traced to the bacterium Enterobacter cloacae, the causal agent of internal yellowing (IY) disease of papaya. Although IY is not discernable on the fruit exterior, internal quality is diminished by fluorescent yellow discolored flesh, tissue softening, and an offensive, rotting odor (Nishijima, 1994; Nishijima et al., 1987).

E. cloacae is widely distributed in the environment (Richard, 1984; Sanders and Sanders, 1997), occurring on or in water, soil, plants, humans, and animals (Richard, 1984). It is also a cross-domain pathogen that causes infections in humans (Sanders and Sanders, 1997) as well as various plant hosts such as elm (Carter, 1945; Murdoch and Campana, 1983), mulberry (Wang et al., 2008), orchid (Takahashi et al., 1997), coconut (George et al., 1976), corn (Rosen, 1922), bulb onion (Bishop and Davis, 1990; Cother and Dowling, 1986), macadamia (Nishijima et al., 2007a), papaya (Nishijima et al., 1987), and mung bean sprouts (Wick et al., 1987). The ability of E. cloacae to cause infections in humans and the occurrence of this bacterium in food crops could pose a food safety risk if contaminated products were ingested in high concentrations or by immune-suppressed individuals.

Internal yellowing disease has been present in Hawaii since the 1980s with incidences in ‘Kapoho Solo’ papaya as high as 43% (Nishijima et al., 1987). Fruit ripeness has been linked to susceptibility to IY, in which disease incidence increases as fruit mature to the full-ripe stage (Nishijima et al., 1987). Disease incidence can also vary with time of year. Surveys conducted from 1985 to 1991 indicated a seasonal variation in IY with higher incidences of disease occurring from October to December (Nishijima, unpublished data). This bacterial disease is an important concern in the food-processing segment of the papaya industry because of the potential risks associated with periods of high IY incidence or seasonal outbreaks and the use of ripe fruit in value-added papaya products. Because coliform bacteria such as E. cloacae can jeopardize food safety of value-added products such as minimally processed fresh or frozen papaya cubes, the use of resistant cultivars could reduce or minimize the occurrence of E. cloacae-infected fruit by having lower incidences of IY. Limited inoculation studies have identified papaya cultivars that are resistant, susceptible, and intermediate to IY infection when challenged with 0.5 mL of E. cloacae at 108 to 109 colony-forming units per milliliter (cfu/mL) inoculum concentration (Nishijima et al., 2004). However, the effects of varying E. cloacae populations on IY response are unknown, including the threshold inoculum concentration necessary to elicit an IY response in susceptible or resistant papaya cultivars.

Papaya cultivars vary in fruit flesh color but can generally be categorized into yellow- or red-fleshed types. Fruit infected with IY have fluorescent yellow discolored tissue that is distinctive in the orange–yellow flesh of ‘Kapoho Solo’ fruit (Nishijima et al., 2004). However, the yellow discoloration is not as apparent in red-fleshed cultivars such as ‘Sunrise’ or ‘SunUp’. The intensity of the yellow color associated with this disease can also vary among some fruit. A colorimeter was used to measure the intensity of grayness in macadamia kernels with gray kernel disease, also caused by E. cloacae (Nishijima et al., 2007a). Similarly, a colorimeter system may be helpful in analyzing IY symptoms of different intensities in yellow- and red-fleshed papaya cultivars. If the colorimeter is more accurate in detecting IY than visual detection, this technology could be applied commercially to screen processed papaya products infected with E. cloacae.

The objectives of this investigation were 1) to determine the effect of various E. cloacae inoculum concentrations on IY incidences and responses of papaya germplasm; 2) to evaluate papaya germplasm for resistance to E. cloacae; and 3) to quantify IY responses among yellow-fleshed and red-fleshed papaya inoculated with E. cloacae using a colorimeter as a method to objectively identify IY-infected papaya tissue.

Materials and Methods

Papaya cultivars, breeding lines, and fruit preparations.

The papaya cultivars and lines selected for evaluation consisted of fruit types exhibiting yellow flesh or red flesh. In addition, some of the evaluated papaya possessed the gene that confers resistance to papaya ringspot virus (PRSV). These genetically engineered (GE) cultivars were Laie Gold F1 (yellow), Rainbow F1 (yellow), and SunUp (red). Also evaluated was I-Rb F5/F6 (yellow), a mixture of two generations (F5 and F6) that were developed as a yellow-fleshed, homozygous PRSV-resistant inbred line derived from ‘Rainbow’. The non-GE cultivars were Kapoho Solo (yellow) and Sunrise (red). These six papaya groups were evaluated in dose–response studies that assessed papaya cultivars and lines for E. cloacae resistance. Four additional papaya groups (all GE) consisting of red-fleshed and yellow-fleshed segregating lines of ‘Rainbow’ F2 and ‘Laie Gold’ F2 were used in a separate study in which colorimeter analyses of infected papaya tissue were conducted to determine if the bright yellow coloration characteristic of IY could be quantitatively distinguished from healthy papaya tissue. Fruit surface area with one-third to half yellow skin color, which indicates papaya at one-third to half ripeness, was obtained from the islands of Oahu, Kauai, and Hawaii. The fruit were washed with cloth gloves and tap water to remove soil and residue on the fruit surface and were air-dried. Before inoculation, the entire fruit surface was disinfected by wiping with 70% ethanol. Each fruit was labeled or marked to identify inoculation dose treatment, cultivar, and inoculation sites.

Bacterial strains and fruit inoculations.

The E. cloacae strains used in dose–response studies consisted of YPV-5B (papaya strain) and ATCC 13047 (type strain from human). Colorimeter analysis studies included two additional strains, B193-3 (ginger strain) and Dd-18 (oriental fruit fly strain). The bacterial strains were grown for 3 to 4 d at 30 °C on PT-M4 agar medium (1.8% bacto-agar, 1% peptone, 0.5% yeast extract, 0.25% sodium chloride, and 0.001% triphenyltetrazolium chloride that was added to sterile, molten agar before pouring) (Nishijima et al., 2004, 2007a). All strains were described or used in previous investigations (Nishijima et al., 1987, 2004, 2007a).

In dose–response studies, inoculum suspensions of E. cloacae strains were prepared by scraping cells from the bacterial cultures, mixing into 40 mL sterile distilled water (SDW), and adjusting the suspension to optical density 0.7 to 0.8 (A600 nm) (1012 cfu/mL) using a Turner SP-830 spectrophotometer (Barnstead/Thermolyne, Dubuque, IA). The concentrated suspensions were serially diluted (1:10 series) into 36 mL SDW (in flasks) to produce bacterial concentrations ranging from 101 to 1012 cfu/mL. All dose treatments were spotted (25-μL aliquots) in duplicate onto PT-M4 plates, incubated overnight at 30 °C, and colonies counted to determine actual bacterial populations of each dilution treatment. For each papaya cultivar or line, at least five fruit of each dose concentration range (0, 101 to 102, 103 to 104, 105 to 106, 107 to 108, 109 to 1010, or 1011 to 1012 cfu/mL) were inoculated in each replicate test. Each test was conducted at least twice. Four sites located along the lengthwise surface of each fruit, one site per corner of an approximate “square grid” at midfruit, were injected (≈1.0-cm depth, 0.5-mL volume) with bacterial inoculum at two sites for each strain or with SDW (control treatment) at all four sites using a sterile 3-mL syringe fitted with a 23-gauge needle. All inoculation sites were covered with tape to prevent cross-contamination from other treatments. Fruit were incubated in fiberboard cartons for 3 d at 26 °C until ripe, dissected at the inoculation sites, and then evaluated for internal yellowing symptoms.

In colorimeter analysis studies of IY responses, fruit were inoculated (0.5 mL) with four E. cloacae strains and SDW control at separate sites along the lengthwise surface of the fruit, similar to methods described previously but with the fifth inoculation site located at the center of the “square grid” of inoculation sites. The inoculum for each strain consisted of a 30-mL aqueous cell suspension that was adjusted to optical density of 0.4 to 0.5 (A600 nm) and equivalent to a bacterial concentration of 108 to 109 cfu/mL (Nishijima et al., 2007a). All inoculation sites on each fruit were covered with tape to prevent cross-contamination from other treatments, and fruit were stored as described previously.

Fruit evaluations.

Ripened fruit were cut open with a flame-sterilized knife, evaluated for IY symptoms by examining tissue dissected at the inoculation sites, and IY incidence and severity responses were determined. IY incidence for fruits was determined by the number of fruit having at least one inoculation site positive for IY out of the total number of fruits, whereas IY incidence for sites was determined by the number sites with positive IY reactions out of the total number of inoculated sites per treatment or IY severity response category. Data for IY incidences in colorimeter studies, along with results of a separate but similar inoculation screening study of the same papaya groups, were used in papaya evaluations for IY resistance that were separate from dose–response studies. IY symptoms were rated for severity responses based on the approximate area at the inoculation site that was affected with distinct yellow discoloration with well-defined or diffuse margins and were categorized as follows: IY0 = no yellowing reaction, IY1w = weak or faint, IY1 = slight (discoloration less than 50% of the inoculation area), IY2 = medium (discoloration 50% to 100% of the inoculation area), and IY3 = severe (discoloration spread beyond the inoculation area). In the dose–response studies, response category IY1w was combined with IY1 category and IY response groups were: IY0, IY1, IY2, and IY3, with IY1 describing weak to slight symptoms. IY responses were analyzed in the colorimeter studies using a Minolta CR-300 chromometer (Minolta Corp., Ramsey, NJ). Hue angle (H°), which quantifies color in the L* C* H color space where 0° = red, 90° = yellow, 180° = green, and 270° = blue, was used to analyze the color of IY-positive (IY1w to IY3) or IY-negative (IY0) papaya tissue that represented symptomatic or non-symptomatic visual observations, respectively.

Reisolation of E. cloacae and determination of bacterial populations.

Putative E. cloacae strains were re-isolated from infected papaya tissues in dose–response studies to confirm the presence of the bacterium in inoculated fruit of various dose treatments and to determine bacterial populations of various IY reactions in resistant or susceptible cultivars or lines. Selected tissue sections (≈1.0 × 1.5 × 0.5 cm) were aseptically excised with a sterile scalpel, weighed, surface-disinfected in 0.5% sodium hypochlorite (plus one to two drops of liquid detergent) (Contrex AL; Decon Laboratories, Inc., King of Prussia, PA) solution for 30 s, drained on clean laboratory tissue, and macerated in 5 mL SDW in a pulsifier bag (Microbiology International, Frederick, MD). An aliquot (1 mL) of the suspension was serially diluted (1:10 series) in 9 mL of SDW in tubes to obtain dilution concentrations ranging from 10−1 to 10−9. All dilution concentrations were spotted (25-μL aliquots in duplicate) onto PT-M4 plates and incubated overnight at 30 °C. Colonies were counted and bacterial populations were calculated as cfu per gram of tissue sample. (Colony-forming units measure viable bacterial cells and are used in microbiology to determine microbial load or concentration.) To confirm the presence of E. cloacae in tissue isolations, individual colonies were isolated, sequentially re-streaked on PT-M4 plates to obtain purified strains, and then tested for oxidative and fermentative reactions in tubes of OF media (Difco Laboratories, Detroit, MI) with glucose as the carbohydrate source. Selected facultative anaerobes were identified according to the manufacturer's instructions using API 20E strips (bioMerieux Inc., Durham, NC) incubated at 30 °C for 18 to 24 h.

To fulfill Koch's postulates in the colorimeter analysis studies, selected tissue sections from IY reactions were analyzed for presence of E. cloacae. Tissues were dissected aseptically and surface-disinfected as described previously. A 1.0-g portion of tissue was macerated aseptically with a sterile glass rod in a test tube containing 9 mL SDW and a 20-μL aliquot of the suspension was streaked onto PT-M4 plates, which were incubated overnight at 30 °C. Methods to confirm E. cloacae among selected purified strains obtained from single colony isolations were performed as described previously.

Naturally occurring internal yellowing disease.

Evaluation of IY symptoms also was conducted for naturally occurring infections among 129 fruit in 2005, 140 fruit in 2006, and 314 fruit in 2007 that were collected from packinghouses or fields on the island of Hawaii. Surveyed cultivars consisted of Kapoho Solo, Rainbow, Sunrise, and SunUp. Fruit were ripened, dissected, and data were collected for IY incidence, IY severity responses, colorimeter analysis of IY symptoms, and bacterial populations, as described previously.

Experimental design and statistical analysis.

The experimental design for the dose–response studies was a factorial with cultivar, inoculum dose, and bacterial treatment (E. cloacae strains ATCC 13047 or YPV-5B, or SDW) factors. A replicate sample consisted of five fruit per inoculum dose treatment, each fruit with four inoculation sites (two per bacterial strain or four SDW). The experiment (all dose treatments) was repeated at least once for each papaya cultivar or line. In colorimeter studies, color analysis was conducted on inoculated fruit with positive (i.e., IY1w, IY1, IY2, or IY3) or negative (IY0) symptoms to quantify visual IY responses in red-fleshed or yellow-fleshed papaya cultivars or lines. The experiment was arranged in a split plot design in which the main plot was fruit flesh color (red or yellow) and the subplots were papaya cultivar or line and inoculation treatment (E. cloacae strain YPV-5B, ATCC 13047, B193-3, or Dd-18; or SDW control). A replicate consisted of at least two individual fruit, each inoculated with all four bacterial strains and SDW control. There were at least four replicates per IY response per fruit flesh color category. Data consisted of hue angle (H°) measurements and hue color category (e.g., yellow or yellow–red). All data (percent IY incidence for fruit or inoculation sites; hue angle) were analyzed by the general linear model procedure of SAS Version 9.2 (SAS Institute, Inc., Cary, NC). Means separation (where appropriate) were performed by pairwise comparisons of means by Fisher's protected least significant difference test at P = 0.05. Linear regression analysis of percent IY incidence means and dose treatments (Log10 cfu/mL) were performed using the regression procedure of SAS (Proc Reg of SAS, Version 9.2).

Results

Inoculum dose–response studies.

The effect of various inoculum concentrations (dose treatments) on IY incidences was used to evaluate papaya germplasm for IY resistance. Dose treatments were presented either as continuous data (Log10 cfu/mL) (e.g., 0, 1, 2, 3, etc.) or as discrete data (Log10 categories cfu/mL) (e.g., 1 to 2, 3 to 4, etc., until 11 to 12). Linear regression analysis indicated that IY incidence (y) could be predicted from inoculum concentration, Log10 cfu/mL (x), for ‘Kapoho Solo’ (y = 8.825x – 8.785; r2 = 0.779; n = 24) (P < 0.0001) and for ‘Laie Gold’ (y = 5.426x – 4.687; r2 = 0.684; n = 24) (P < 0.0001), but not for ‘Rainbow’, I-Rb F5/F6, ‘SunUp’, or ‘Sunrise’ (r2 = 0.067, 0.049, 0.092, 0.028, respectively) (all P > 0.05). Also, the relationship of IY incidences and increasing dose treatments of E. cloacae was positively correlated for susceptible cultivars Kapoho Solo (r = 0.883) (P < 0.0001) and Laie Gold (r = 0.827) (P < 0.0001) but not for resistant cultivars or lines (P > 0.05).

Analysis of variance of IY incidence data indicated significant differences among means for cultivar and inoculum dose category (Log10 category, cfu/mL) but not for bacterial strain (P < 0.0001, P < 0.0001, P = 0.21, respectively) (Table 1). Interactions were significant only for cultivar × dose category (P < 0.0001). ‘Kapoho Solo’ had the highest IY incidence (44.2%) among six papaya cultivars or lines, whereas dose treatment categories 7 to 8, 9 to 10, and 11 to 12 Log10 cfu/mL produced IY incidences of 23.5%, 25.8%, and 30.0%, respectively (Table 1). When data were analyzed by dose treatment category, means for cultivars were significantly different at dose treatment category 9 to 10 Log10 (P = 0.03) and 11 to 12 Log10 (P = 0.05) but were not different at the other dose treatment categories (P = 0.33 to 0.46) (analysis not shown). The two most susceptible cultivars were differentiated at dose treatment categories 9 to 10 and 11 to 12 Log10 with ‘Kapoho Solo’ having the highest IY incidences (90.0% and 87.5%, respectively) followed by ‘Laie Gold’ F1 (50.0% and 52.5%, respectively) (Table 2). Mean IY incidences among the other four cultivars were no higher than 26.7% (Table 2), indicating some level of resistance to the pathogen. When data were sorted and analyzed by cultivar, the effect for dose treatment category was significant only for ‘Kapoho Solo’ (P = 0.006) and ‘Laie Gold’ F1 (P = 0.013). The results were similar to findings in the colorimeter papaya screening study in which red- or yellow-fleshed papaya were inoculated with E. cloacae strains (ATCC 13047, B193-3, Dd-18, or YPV-5B) at 109 cfu/mL inoculum concentration and were evaluated for IY symptoms and color-analyzed with a colorimeter (data shown in Table 3). Papaya groups were categorized as resistant (10% or less IY), moderately resistant (11% to 39% IY), moderately susceptible (40% to 59% IY), or susceptible (60% or greater IY) based on IY incidence data gathered in this study. Using these resistance-susceptible categories and corresponding disease incidence criteria, yellow-fleshed cultivars Kapoho Solo and Laie Gold F1 were susceptible (81.3% and 65.2% IY, respectively), I-Rb F5/F6 and ‘Rainbow’ F1 were moderately resistant (16.8% and 33.1% IY, respectively), and red-fleshed cultivars Sunrise and SunUp were resistant (0% and 3.6% IY, respectively) (Table 4).

Table 1.

Effects of papaya cultivar or line, Enterobacter cloacae strain (ATCC 13047 or YPV-5B), and inoculum concentration (Log10 category cfu/mL) on mean percent internal yellowing (IY) incidence of inoculated sites.

Table 1.
Table 2.

Mean percent internal yellowing (IY) incidence in yellow- or red-fleshed papaya cultivars or lines inoculated with E. cloacae strains (ATCC 13047 or YPV-5B) at various inoculum concentrations.

Table 2.
Table 3.

Effect of cultivar or line, inoculation treatment, internal yellowing (IY) response, and hue color on hue angle values of red- or yellow-fleshed papaya cultivars or lines inoculated with Enterobacter cloacae strains (ATCC 13047, B193-3, Dd-18, or YPV-5B) or sterile distilled water (SDW).

Table 3.
Table 4.

Evaluation of red- or yellow-fleshed papaya cultivars and lines for resistance to internal yellowing (IY) infection in fruits inoculated with Enterobacter cloacae strains (ATCC 13047, B193-3, Dd-18, or YPV-5B) at 109 cfu/mL.

Table 4.

The lowest dose category tested for ‘Kapoho Solo’ and ‘Laie Gold’ F1 was at 3 to 4 Log10 cfu/mL, which elicited 12.5% and 6% IY incidence, respectively, and represents an approximate minimum bacterial population necessary for symptom expression in the most susceptible cultivars (Table 2). For ‘Laie Gold’ F1, dose treatment categories greater than 7 to 8 Log10 cfu/mL had an apparent “plateau effect” in which, despite increasing inoculum concentrations, mean percent IY incidence stabilized at 50.0% to 52.5% (Table 2), indicating that this cultivar was less susceptible than ‘Kapoho Solo’. Minimum inoculum concentrations necessary to elicit an IY response varied for the other cultivars with resistant cultivar Sunrise producing IY symptoms only at 9 to 10 Log10 cfu/mL, whereas moderately resistant to resistant papayas I-Rb F5/F6, ‘Rainbow’ F1, and ‘SunUp’ produced IY symptoms at doses 1 to 2 and 3 to 4 Log10 cfu/mL (Table 2).

In evaluations of IY severity responses resulting from papaya inoculations at various dose treatment categories, ‘Kapoho Solo’ was the only cultivar among the evaluated cultivars and lines that exhibited increasing incidences of severe IY3 reactions with increasing inoculum concentrations (Fig. 1). There were also proportionately greater incidences of IY3 response (57%) than the less severe IY1 and IY2 responses (10% and 23%, respectively) when inoculated at concentration 9 to 10 Log10 cfu/mL (Fig. 1). There were no occurrences of IY3 responses in the other papaya groups at this inoculation concentration (data not shown). These results confirmed that ‘Kapoho Solo’ is the most susceptible cultivar and inoculum concentration 9 to 10 Log10 cfu/mL is optimal for evaluating papaya for resistance to E. cloacae (Table 2).

Fig. 1.
Fig. 1.

Incidence of internal yellowing (IY) severity responses of ‘Kapoho Solo’ papayas inoculated with Enterobacter cloacae strains (ATCC 13047 or YPV-5B) at varying inoculum concentrations (range Log10, cfu/mL). Vertical bars represent SEM.

Citation: HortScience horts 45, 9; 10.21273/HORTSCI.45.9.1357

Colorimeter studies.

For the 10 papaya groups that were evaluated, hue angle (H°) means between papaya of red or yellow flesh color were significantly different (P = 0.003) and data were sorted into separate flesh color data sets for further analysis. Analysis of variance for red-fleshed papaya indicated significant differences among hue angle means for cultivar or line (P = 0.005) and hue color category (P = 0.040). Yellow-fleshed papaya had significant differences in means for IY response (P = 0.0004) and hue color category (P < 0.0001). Among four red-fleshed cultivars or lines, a red-fleshed segregant of ‘Laie Gold’ F2 had the highest hue angle mean (72.1°), whereas ‘Sunrise’ was lowest (59.4°) (Table 3). Among yellow-fleshed cultivars or lines, hue angle means were 75.6 to 78.8° and were not significantly different (P = 0.695). Interestingly, hue angle means for yellow-fleshed segregants of ‘Laie Gold’ F2 and ‘Rainbow’ F2 were similar in values to their yellow-fleshed F1 counterparts. However, hue angle mean of red-fleshed segregant of ‘Laie Gold’ F2 was significantly higher (72.1°) than means for the other red-fleshed papaya groups (59.4, 61.6, 64.7°) and indicates more “yellowness” in this papaya group (Table 3).

Among IY responses, hue angle means of symptomatic IY reactions (IY1w to IY3) were significantly higher than non-symptomatic reaction (IY0) for yellow-fleshed papaya groups (Table 3) and indicated that infected tissue can be distinguished from non-infected tissue. A similar but not significant (P = 0.089) trend was observed for red-fleshed papayas. When data for symptomatic responses were analyzed by individual papaya groups, red-fleshed papaya ‘Sun-Up’, ‘Sunrise’, and ‘Rainbow’ F2-red segregant had the lowest hue angle (H°) means (66.2, 69.7, and 74.7°, respectively), whereas all other red- or yellow-fleshed papaya groups had hue angle means of 77 to 81° (data not shown). The same red-fleshed cultivars also had the lowest hue angle means (61.1, 59.4, and 63.4°, respectively) in non-symptomatic responses (data not shown). Hue angle means among IY severity responses for papaya groups were also evaluated. When data were sorted by individual cultivar or line, only yellow-fleshed ‘Kapoho Solo’ had hue angle means (73.6, 76.1, 78.5, 79.4, and 80.9°) that were significantly different (P = 0.039) as well as positively related to increasing IY severity responses (IY0, IY1w, IY1, IY2, and IY3, respectively) (Table 5). Other yellow-fleshed papaya groups with hue angle means that were significantly different among IY severity responses were ‘Rainbow’ F1 (P = 0.017) and ‘Laie Gold’ F2-yellow segregant (P = 0.035) (Table 5).

Table 5.

Mean hue angle values of internal yellowing (IY) responses of red- or yellow-fleshed papaya cultivars or lines inoculated with Enterobacter cloacae strains (ATCC 13047, B193-3, Dd-18, or YPV-5B) or sterile distilled water (SDW).

Table 5.

Colorimeter readings were recorded as hue angle values and the corresponding hue color category (Y = yellow; YR = yellow–red) that is assigned by the colorimeter. The hue angle means were significantly higher for Y (80.1 and 80.7°) than YR (63.1 and 75.2°) in both papaya flesh groups (red and yellow, respectively) (Table 3), cultivars, or lines (data not shown) and all IY response categories (IY0, IY1w, IY1, IY2, and IY3) (data not shown). Additionally, the hue angle means (80° and higher) for inoculation reactions of Y hue color were not significantly different (P > 0.05) between red- and yellow-fleshed papaya groups or between cultivars or lines, indicating that infected papaya with hue angle values 80° or greater and designated as Y hue color denote a positive IY reaction in red- or yellow-fleshed papaya. This criterion would be useful in distinguishing infected and non-infected papaya tissue in a breeding program.

To further analyze the accuracy of using Y hue color designation as an indicator of IY infection, the percent incidence of Y hue color among symptomatic or non-symptomatic IY responses was determined and used to compare visual designation of IY positive or negative reactions, respectively, to colorimeter analysis (Y hue color). For symptomatic reactions, red-fleshed papaya inoculated with E. cloacae strains resulted in lower percent incidences of IY occurrence among inoculated sites than yellow-fleshed papaya and ranged from 1.3% (‘Sunrise’) to 25.7% (‘Laie Gold’ F2-red segregant) compared with 31.9% (‘Rainbow’ F1) to 82.1% (‘Rainbow’ F2-yellow segregant), respectively (data presented as number of occurrences; Table 6). Symptomatic IY that was confirmed by Y hue color among red-fleshed papaya was in red segregants of ‘Laie Gold’ F2 and ‘Rainbow’ F2, which had Y hue color occurrences of 79.0% and 33.3%, respectively (data presented as number of occurrences; Table 6). The 67% visual IY positive that was not Y hue color (i.e., “false” IY positive) that was encountered in ‘Rainbow’ F2-red segregant exemplifies the occasional difficulty of visual designation of IY in red-fleshed papayas. Among yellow-fleshed papaya, ‘Rainbow’ F1 and yellow segregant of ‘Laie Gold’ F2 had the highest incidences of symptomatic IY responses that were confirmed by Y hue color, 91.3% and 86.2%, respectively (data presented as number of occurrences; Table 6). Among papaya with non-symptomatic IY responses, the percent incidence of Y hue color was 0% or at most 14.3%, which was in the yellow segregant of ‘Rainbow’ F2 (data presented as number of occurrences; Table 6). Colorimeter analysis possibly detected positive IY responses that were not visibly observed in the yellow-fleshed segregant of ‘Rainbow’ F2, which had among the highest H° means (and therefore “yellower” flesh) among the papaya cultivars or lines (Table 6). The accuracy of Y hue color relative to visually positive IY reaction was also evaluated by determining the percent incidence of symptomatic IY response among Y hue color designations. Results were similar for red- and yellow-fleshed papaya in which 91% to 100% of bacteria-inoculated sites that were Y hue color by colorimeter also were visually IY positive (Table 6). Hue angle means for papaya groups with Y hue color designations and visually symptomatic tissue ranged from 80 to 82°, except for 78.6° in a red-fleshed papaya (‘Rainbow’ F2-red segregant) (Table 6). These results demonstrate the usefulness of Y hue color in confirming IY-positive reactions in red- or yellow-fleshed papaya.

Table 6.

Comparison of visual designations with colorimeter analysis for hue Y (yellow) color for non-symptomatic or symptomatic internal yellowing (IY) responses of red- or yellow-fleshed papaya cultivars or lines inoculated with Enterobacter cloacae strains (ATCC 13047, B193-3, Dd-18, or YPV-5B).z

Table 6.

Reisolation of E. cloacae and determination of bacterial populations.

IY severity response categories were not correlated with final bacterial concentration (data not shown). However, when individual dose treatments were sorted into two categories (101 to 108 or 109 to 1012 cfu/mL) for data analysis, inoculum concentration affected final bacterial concentration of overall IY responses. Final bacterial populations (ranging from 106 to 109 cfu/mL) increased by ≈3 to 5 Log10 cfu/g when low to medium doses (101 to 108 cfu/mL) were used to inoculate five cultivars (Kapoho Solo, Laie Gold F1, Rainbow F1, Sunrise, SunUp) or one line (I-Rb F5/F6) in dose–response studies (data not shown). In contrast, final bacterial populations (ranging from 107 to 1010 cfu/mL) decreased by ≈1 to 2 Log10 cfu/g when high dose inoculum treatments (109 to 1012 cfu/mL) were used (data not shown). The high dose range may represent a saturation range for E. cloacae inoculation. Re-isolated bacterial strains from each papaya cultivar or line in dose–response studies and colorimeter studies were repeatedly identified as E. cloacae according to procedures described earlier, thus fulfilling Koch's postulates (data not shown). E. cloacae was also confirmed in red- and yellow-fleshed papaya with symptomatic or weak or non-symptomatic IY responses that were Y hue color.

Naturally occurring internal yellowing.

Fruit evaluated for IY from collections on Hawaii island during 2005 to 2007 showed naturally occurring IY of severity categories ranging from IY1 to IY3 in six fruit of ‘Rainbow’ F1, six fruit of ‘Kapoho Solo’, and two fruit of ‘Laie Gold’ F2 (data not shown). When infected tissues were analyzed using the colorimeter, all tissues were Y hue color with a mean H° of 81.9. These results were comparable to those for inoculated yellow-fleshed papaya (n = 194) in which the mean H° was 80.7 for tissue of Y hue color (Table 3). Naturally occurring IY incidences varied for the four surveyed cultivars and ranged from 2.6% IY in ‘Rainbow’ F1 (seven of 269 fruit) to 12.7% IY in ‘Kapoho Solo’ (30 of 237 fruit). There was no incidence of IY among surveyed fruit of ‘Sunrise’ (39 fruit) or ‘SunUp’ (38 fruit). ‘Kapoho Solo’ fruit evaluated with IY symptoms were confirmed to be infected with E. cloacae with bacterial populations ranging from 7.4 × 103 to 8.57 × 107 cfu/g; however, E. cloacae was not isolated from ‘Rainbow’ F1 fruit with IY symptoms.

Discussion

Internal yellowing, caused by E. cloacae, is a limiting factor for production of value-added products such as fresh or frozen papaya cubes. High coliform counts and reduced quality as a result of yellow discoloration and tissue softening are factors that can lead to product rejection. Identifying or developing IY-resistant papaya cultivars can help reduce the risk of bacterial contamination and ensure compliance with food safety regulations. We evaluated papaya cultivars and lines for IY resistance and identified an optimal inoculum concentration of 9 to 10 Log10 cfu/mL that differentiated resistant and susceptible germplasm. Based on IY incidence ranges as criteria, the six evaluated cultivars or lines were ranked for resistance at this dose concentration as follows: ‘Sunrise’, ‘SunUp’, and I-Rb F5/F6 (resistant, 10% or less IY); ‘Rainbow’ F1 (moderately resistant, 11% to 39% IY); ‘Laie Gold’ F1 (moderately susceptible, 40% to 59% IY); and ‘Kapoho Solo’ (susceptible, 60% or greater IY). These six cultivars or lines were similarly ranked in colorimeter studies in which positive IY incidence data were used. Two resistant cultivars (Sunrise and SunUp, both red-fleshed papaya), four moderately resistant cultivars or lines (red-fleshed segregants of ‘Rainbow’ F2 and ‘Laie Gold’ F2 and yellow-fleshed I-Rb F5/F6 and ‘Rainbow’ F1). and two moderately susceptible lines (yellow-fleshed segregants of ‘Laie Gold’ F2 and ‘Rainbow’ F2) were identified (Table 4). The highly susceptible nature of ‘Kapoho Solo’ (Nishijima et al., 2004) was confirmed (Table 4). ‘Laie Gold’ F1 and I-Rb F5/F6 had higher IY incidences in the colorimeter studies than in dose–response studies and were determined susceptible and moderately resistant, respectively (Table 4), rather than moderately susceptible and resistant.

Four of the five most resistant papaya groups evaluated were red-fleshed papaya (Table 4) and indicate a possible natural resistance to IY in this papaya flesh color group. Similarly, evaluation of onion bulb cultivars indicated that red onion bulb cultivars were more resistant than white and yellow cultivars when inoculated with E. cloacae (Schroeder et al., 2010) and imply that disease resistance factors such as pigments or phenolic substances may be associated with red-pigmented plants (Gandikota et al., 2001; Link, et al., 1929; Link and Walker, 1933). The most resistant yellow-fleshed papaya group was I-Rb F5/F6, which represents the best potential for development of resistant yellow-fleshed papayas and merits further evaluation. Disease resistance factors in this papaya line may be attributed to its red flesh genetic heritage through its ‘SunUp’ lineage.

Our studies also determined that the bright yellow coloration of IY-infected fruit can be measured with a colorimeter and that infected tissue can be distinguished from healthy papaya tissue in red- or yellow-fleshed fruit. Infected tissue that was visibly symptomatic usually had Y hue color with 79.1 to 81.1° hue angle means among red- or yellow-fleshed papayas. Additionally, the incidence of inoculation sites with Y hue color that were also symptomatic for IY ranged from 91% (‘Rainbow’ F1) to 100% (‘Rainbow’ F2-red segregant, I-Rb F5/F6, ‘Laie Gold’ F1, and ‘Kapoho Solo’) and demonstrates the accuracy of this color analysis element in identifying infected tissues. In comparison, YR hue color occurred at 86.7% incidence in non-symptomatic tissue, resulted in significantly lower (P = 0.002) hue angle means for red-fleshed papaya compared with yellow-fleshed papaya (63.1° and 75.1°, respectively), and indicates an association with non-infected tissue. These results demonstrate the capability of color analysis in papaya breeding programs for evaluating resistance to IY or in commercial operations for sorting infected from non-infected fruit and fruit products.

Although fruit inoculations with the optimal dose treatment of 9 to 10 Log10 cfu/mL was effective in evaluating papaya cultivars for resistance to E. cloacae infection, the varying dose–response protocol used in this study also was valuable in producing dose–response profiles for cultivars or lines and demonstrated the unpredictable nature of cultivar evaluations. The dose–response profile for ‘SunUp’ revealed unexpected results for IY incidences at different dose treatments (e.g., 5 to 6 and 7 to 8 Log10 cfu/mL) (Table 2) and contradicted an earlier evaluation of this cultivar as immune (Nishijima et al., 2004).

Bacterial contamination in papaya by E. cloacae (Nishijima et al., 1987) or E. sakazakii (= Cronobacter sakazakii) (Keith et al., 2008) from naturally occurring infections in papaya fruit poses a food safety risk and represents an obstacle to the development of fresh-cut or frozen papaya products. Our studies indicated that E. cloacae populations can occur as high as 8.57 × 107 cfu/g in naturally infected papaya, which is higher than a previously reported population of 4.17 × 105 cfu/g (Nishijima et al., 2007b). Both populations, occurring in ‘Kapoho Solo’ fruit, exceed the food safety guideline limit for coliforms of less than 100 cfu/g (Sodexho Supplier Code of Practice, 2007) that is being used by individuals in the papaya industry. Although the minimum E. cloacae population required to elicit infection in humans is not known, it is possible that this cross-domain (i.e., plant and human) pathogen can pose a risk to susceptible individuals through contaminated vegetables and fruit (Neto et al., 2003).

In our surveys of papaya with naturally occurring IY, we were not able to isolate E. cloacae from ‘Rainbow’ F1 fruit. The occasional non-culturable nature of this bacterium was observed previously with gray kernel disease of macadamia (Nishijima et al., 2007a).

Specific food technology methods and techniques are needed to treat papaya and papaya products to ensure a safe and high-quality commodity. Non-thermal technologies (e.g., high hydrostatic pressure, pulsed electric fields, ionizing radiation, ultrasonication) (Ross et al., 2003), vacuum and steam technology (Kozempel et al., 2002), and traditional and alternative antimicrobial treatments (Raybaudi-Massilia et al., 2009) are examples of technologies used to control pathogenic and spoilage microorganisms in fresh-cut fruits and vegetables. Promising technologies for preserving quality and preventing deterioration of fresh-cut papaya or frozen papaya cubes include applications of chitosan coating (Gonzalez-Aguilar et al., 2009) or combination treatments of ozone wash and heat that reduce E. cloacae microbial counts and enhance papaya quality and flavor (Yonemura, 2009). The baseline data of natural populations of E. cloacae in papaya that were generated in this investigation could be useful in dose–response or kinetics profiles of the various food treatment systems.

The role of fruit flies and other insect foragers in the transmission of human diseases through contaminated food is an emerging area of interest (Janisiewicz et al., 1999; Sela et al., 2005) that may provide insight into the possible mode of entry of E. cloacae in papaya fruit. E. cloacae occurs in the stomach and gut of the oriental fruit fly (Bactrocera dorsalis Hendel) (Jang and Nishijima, 1990), and papaya is a host of tephritid fruit flies, including B. dorsalis (Liquido et al., 1989). The occurrence of E. cloacae in internal yellowing-diseased papaya fruit and in washes of papaya flowers supports the hypothesis that fruit flies could transmit this bacterial plant pathogen to papayas (Nishijima et al., 1987). The preferential status of different papaya cultivars for fruit fly insect pests is an area that may be worthy of investigation as a way to select papaya germplasm that are less susceptible to fruit fly infestation and have less potential for high bacterial populations in fruit.

In conclusion, our protocol for treating papaya cultivars with various inoculum concentrations produced incidence and severity response profiles relative to bacterial concentration that were useful in evaluations for IY resistance. Minimum, maximum, and optimum dose concentrations were identified and used to rank papaya cultivars and lines for resistance. Naturally occurring populations of E. cloacae in ‘Kapoho Solo’ were identified that were higher than previous reports and underscored the importance of finding food processing or horticultural methods (e.g., breeding for resistance) to minimize microbial contamination in papaya. The use of a colorimeter to identify infected papaya fruit was demonstrated in which hue angle ranges and Y hue color characterized IY-infected tissue in red- and yellow-fleshed papayas. The application of this technology as well as other food processing technologies and the use of resistant papaya cultivars would help encourage market growth in fresh-cut or frozen papaya cube products by ensuring a safe food product.

Literature Cited

  • Bishop, A.L. & Davis, R.M. 1990 Internal decay of onions caused by Enterobacter cloacae Plant Dis. 74 692 694

  • Carter, J.C. 1945 Wetwood of elms Ill. Nat. Hist. Surv. Bull. 23 407 448

  • Cother, E.J. & Dowling, V. 1986 Bacteria associated with internal breakdown of onion bulbs and their possible role in disease expression Plant Pathol. 35 329 336

    • Search Google Scholar
    • Export Citation
  • Gandikota, M., de Kochko, A., Chen, L., Ithal, N., Fauquet, C. & Reddy, A.R. 2001 Development of transgenic rice plants expressing maize anthocyanin genes and increased blast resistance Mol. Breed. 7 73 83

    • Search Google Scholar
    • Export Citation
  • George, M., Potty, V.P. & Jayasankar, N.P. 1976 Association of Enterobacter with coconut root (wilt) disease Curr. Sci. 45 677 678

  • Gonzalez-Aguilar, G.A., Valenzuela-Soto, E., Lizardi-Mendoza, J., Goycoolea, F., Martinez-Tellez, M.A., Villegas-Ochoa, M.A., Monroy-Garcia, I.N. & Ayala-Zavala, J.F. 2009 Effect of chitosan coating in preventing deterioration and preserving the quality of fresh-cut papaya ‘Maradol’ J. Sci. Food Agr. 89 15 23

    • Search Google Scholar
    • Export Citation
  • Jang, E.B. & Nishijima, K.A. 1990 Identification and attractancy of bacteria associated with Dacus dorsalis (Diptera: Tephritidae) Environ. Entomol. 19 1726 1731

    • Search Google Scholar
    • Export Citation
  • Janisiewicz, W.J., Conway, W.S., Brown, M.W., Sapers, G.M., Fratamico, P. & Buchanan, R.L. 1999 Fate of Escherichia coli O157:H7 on fresh-cut apple tissue and it's potential for transmission by fruit flies Appl. Environ. Microbiol. 65 1 5

    • Search Google Scholar
    • Export Citation
  • Keith, R.C., Nishijima, K.A., Keith, L.M., Fitch, M.M., Nishijima, W.T. & Wall, M.M. 2008 Atypical internal yellowing of papaya fruit in Hawaii caused by Enterobacter sakazakii Plant Dis. 92 487

    • Search Google Scholar
    • Export Citation
  • Kozempel, M., Radewonuk, E.R., Scullen, O.J. & Goldberg, N. 2002 Application of the vacuum/steam/vacuum surface intervention process to reduce bacteria on the surface of fruits and vegetables Innov. Food Sci. Emerg. Technol. 3 63 72

    • Search Google Scholar
    • Export Citation
  • Link, K.P., Dickson, A.D. & Walker, J.C. 1929 Further observations on the occurrence of protocatechuic acid in pigmented onion scales and its relation to disease resistance in the onion J. Biol. Chem. 84 719 725

    • Search Google Scholar
    • Export Citation
  • Link, K.P. & Walker, J.C. 1933 The isolation of catechol from pigmented onion scales and its significance in relation to disease resistance in onions J. Biol. Chem. C 379 383

    • Search Google Scholar
    • Export Citation
  • Liquido, N.J., Cunningham, R.T. & Couey, H.M. 1989 Infestation rates of papaya by fruit flies (Diptera: Tephritidae) in relation to the degree of fruit ripeness J. Econ. Entomol. 82 213 219

    • Search Google Scholar
    • Export Citation
  • Murdoch, C.W. & Campana, R.J. 1983 Bacteria species associated with wetwood of elm Phytopathology 73 1270 1273

  • Neto, J.R., Yano, T., Beriam, L.O.S., Destefano, S.A.L., Oliveira, V.M. & Rosato, Y.B. 2003 Comparative RFLP-ITS analysis between Enterobacter cloacae strains isolated from plants and clinical origin Arq. Inst. Biol. (Sao Paulo) 70 367 372

    • Search Google Scholar
    • Export Citation
  • Nishijima, K.A. 1994 Papaya diseases caused by bacteria. Internal yellowing 65 Ploetz R.C., Zentmyer G.A., Nishijima W.T., Rohrbach K.G. & Ohr H.D. Compendium of tropical fruit diseases The American Phytopathological Society Press St. Paul, MN

    • Search Google Scholar
    • Export Citation
  • Nishijima, K.A., Alvarez, A.M., Hepperly, P.R., Shintaku, M.H., Keith, L.M., Sato, D.M., Bushe, B.C., Armstrong, J.W. & Zee, F.T. 2004 Association of Enterobacter cloacae with rhizome rot of edible ginger in Hawaii Plant Dis. 88 1318 1327

    • Search Google Scholar
    • Export Citation
  • Nishijima, K.A., Couey, H.M. & Alvarez, A.M. 1987 Internal yellowing, a bacterial disease of papaya fruits caused by Enterobacter cloacae Plant Dis. 71 1029 1034

    • Search Google Scholar
    • Export Citation
  • Nishijima, K.A., Wall, M.M. & Siderhurst, M.S. 2007a Demonstrating pathogenicity of Enterobacter cloacae on macadamia and identifying associated volatiles of gray kernel of macadamia in Hawaii Plant Dis. 91 1221 1228

    • Search Google Scholar
    • Export Citation
  • Nishijima, K.A., Keith, R.C., Fitch, M.M., Wall, M.M., Sugiyama, L.S. & Nishijima, W.T. 2007b Production of internal yellowing symptoms on resistant and susceptible papaya cultivars by Enterobacter cloacae at varying inoculum concentrations Phytopathology 97 S84

    • Search Google Scholar
    • Export Citation
  • Raybaudi-Massilia, R.M., Mosqueda-Melgar, J., Soliva-Fortuny, R. & Martin- Belloso, O. 2009 Control of pathogenic and spoilage microorganisms in fresh-cut fruits and fruit juices by traditional and alternative natural antimicrobials Comp. Rev. Food Sci. Food Safety 8 157 180

    • Search Google Scholar
    • Export Citation
  • Richard, C. 1984 Genus VI. Enterobacter Hormaeche and Edwards 1960, 72; Nom. Cons. Opin. 28. Jud. Comm. 1963, 38 465 469 Kreig N.R. & Holt J.G. Bergey's manual of systemic bacteriology Vol. 1 Williams & Wilkins Baltimore, MD

    • Search Google Scholar
    • Export Citation
  • Rosen, H.R. 1922 The bacterial pathogen of corn stalk rot Phytopathology 12 497 499

  • Ross, A.I.V., Griffiths, M.W., Mittal, G.S. & Deeth, H.C. 2003 Combining nonthermal technologies to control foodborne microorganisms Int. J. Food Microbiol. 89 125 138

    • Search Google Scholar
    • Export Citation
  • Sanders, W.E. & Sanders, C.C. 1997 Enterobacter spp.: Pathogens poised to flourish at the turn of the century Clin. Microbiol. Rev. 10 220 241

  • Schroeder, B.K., Waters, T.D. & du Toit, L.J. 2010 Evaluation of onion cultivars for resistance to Enterobacter cloacae in storage Plant Dis. 94 236 243

    • Search Google Scholar
    • Export Citation
  • Sela, S., Nestel, D., Pinto, R., Nemny-Lavyand, E. & Bar-Joseph, M. 2005 Mediterranean fruit fly as a potential vector of bacterial pathogens Appl. Environ. Microbiol. 71 4052 4056

    • Search Google Scholar
    • Export Citation
  • Sodexho Supplier Code of Practice 2007 Fruit & vegetables—Microbiological criteria: Prepared, ready-to-eat and frozen products 71 16 Aug. 2007 <http://uk.sodexo.com/uken/Images/Suppcodesmaller_tcm15-50823.pdf>.

    • Search Google Scholar
    • Export Citation
  • Takahashi, Y., Takahashi, K., Sato, M., Watanabe, K. & Kawano, T. 1997 Bacterial leaf rot of Odontioda orchids caused by Enterobacter cloacae Ann. Phytopathological Soc. Jpn. 63 164 169

    • Search Google Scholar
    • Export Citation
  • Wang, G.F., Praphat, K., Xie, G.L., Zhu, B. & Li, B. 2008 Bacterial wilt of mulberry (Morus alba) caused by Enterobacter cloacae in China Plant Dis. 92 483

    • Search Google Scholar
    • Export Citation
  • Wick, R.L., Rane, K.K. & Sutton, D.K. 1987 Mung bean sprout disease caused by Enterobacter cloacae Phytopathology 77 123 [abstr.].

  • Yonemura, R.K. 2009 Improved method of processing papayas for food safety and quality—IQF Dream, LLC 4 Aug. 2009 <http://www.reeis.usda.gov/web/crisprojectpages/206495.html>.

    • Search Google Scholar
    • Export Citation

Contributor Notes

This research was funded in part by the USDA Special Grants Program for Tropical and Subtropical Agriculture Research.

We gratefully acknowledge Orlando Manuel and Ken Harada for their donations of papaya fruit for our studies, and we appreciate the laboratory assistance of Jean Doherty and Stephanie Weis.

Current address: Hawaii Agriculture Research Center, P.O. Box 100, Kunia, HI 96759.

Current address: USDA, ARS, PBARC, P.O. Box 4459, Hilo, HI 96720.

To whom reprint requests should be addressed; e-mail kate.nishijima@ars.usda.gov.

  • View in gallery

    Incidence of internal yellowing (IY) severity responses of ‘Kapoho Solo’ papayas inoculated with Enterobacter cloacae strains (ATCC 13047 or YPV-5B) at varying inoculum concentrations (range Log10, cfu/mL). Vertical bars represent SEM.

  • Bishop, A.L. & Davis, R.M. 1990 Internal decay of onions caused by Enterobacter cloacae Plant Dis. 74 692 694

  • Carter, J.C. 1945 Wetwood of elms Ill. Nat. Hist. Surv. Bull. 23 407 448

  • Cother, E.J. & Dowling, V. 1986 Bacteria associated with internal breakdown of onion bulbs and their possible role in disease expression Plant Pathol. 35 329 336

    • Search Google Scholar
    • Export Citation
  • Gandikota, M., de Kochko, A., Chen, L., Ithal, N., Fauquet, C. & Reddy, A.R. 2001 Development of transgenic rice plants expressing maize anthocyanin genes and increased blast resistance Mol. Breed. 7 73 83

    • Search Google Scholar
    • Export Citation
  • George, M., Potty, V.P. & Jayasankar, N.P. 1976 Association of Enterobacter with coconut root (wilt) disease Curr. Sci. 45 677 678

  • Gonzalez-Aguilar, G.A., Valenzuela-Soto, E., Lizardi-Mendoza, J., Goycoolea, F., Martinez-Tellez, M.A., Villegas-Ochoa, M.A., Monroy-Garcia, I.N. & Ayala-Zavala, J.F. 2009 Effect of chitosan coating in preventing deterioration and preserving the quality of fresh-cut papaya ‘Maradol’ J. Sci. Food Agr. 89 15 23

    • Search Google Scholar
    • Export Citation
  • Jang, E.B. & Nishijima, K.A. 1990 Identification and attractancy of bacteria associated with Dacus dorsalis (Diptera: Tephritidae) Environ. Entomol. 19 1726 1731

    • Search Google Scholar
    • Export Citation
  • Janisiewicz, W.J., Conway, W.S., Brown, M.W., Sapers, G.M., Fratamico, P. & Buchanan, R.L. 1999 Fate of Escherichia coli O157:H7 on fresh-cut apple tissue and it's potential for transmission by fruit flies Appl. Environ. Microbiol. 65 1 5

    • Search Google Scholar
    • Export Citation
  • Keith, R.C., Nishijima, K.A., Keith, L.M., Fitch, M.M., Nishijima, W.T. & Wall, M.M. 2008 Atypical internal yellowing of papaya fruit in Hawaii caused by Enterobacter sakazakii Plant Dis. 92 487

    • Search Google Scholar
    • Export Citation
  • Kozempel, M., Radewonuk, E.R., Scullen, O.J. & Goldberg, N. 2002 Application of the vacuum/steam/vacuum surface intervention process to reduce bacteria on the surface of fruits and vegetables Innov. Food Sci. Emerg. Technol. 3 63 72

    • Search Google Scholar
    • Export Citation
  • Link, K.P., Dickson, A.D. & Walker, J.C. 1929 Further observations on the occurrence of protocatechuic acid in pigmented onion scales and its relation to disease resistance in the onion J. Biol. Chem. 84 719 725

    • Search Google Scholar
    • Export Citation
  • Link, K.P. & Walker, J.C. 1933 The isolation of catechol from pigmented onion scales and its significance in relation to disease resistance in onions J. Biol. Chem. C 379 383

    • Search Google Scholar
    • Export Citation
  • Liquido, N.J., Cunningham, R.T. & Couey, H.M. 1989 Infestation rates of papaya by fruit flies (Diptera: Tephritidae) in relation to the degree of fruit ripeness J. Econ. Entomol. 82 213 219

    • Search Google Scholar
    • Export Citation
  • Murdoch, C.W. & Campana, R.J. 1983 Bacteria species associated with wetwood of elm Phytopathology 73 1270 1273

  • Neto, J.R., Yano, T., Beriam, L.O.S., Destefano, S.A.L., Oliveira, V.M. & Rosato, Y.B. 2003 Comparative RFLP-ITS analysis between Enterobacter cloacae strains isolated from plants and clinical origin Arq. Inst. Biol. (Sao Paulo) 70 367 372

    • Search Google Scholar
    • Export Citation
  • Nishijima, K.A. 1994 Papaya diseases caused by bacteria. Internal yellowing 65 Ploetz R.C., Zentmyer G.A., Nishijima W.T., Rohrbach K.G. & Ohr H.D. Compendium of tropical fruit diseases The American Phytopathological Society Press St. Paul, MN

    • Search Google Scholar
    • Export Citation
  • Nishijima, K.A., Alvarez, A.M., Hepperly, P.R., Shintaku, M.H., Keith, L.M., Sato, D.M., Bushe, B.C., Armstrong, J.W. & Zee, F.T. 2004 Association of Enterobacter cloacae with rhizome rot of edible ginger in Hawaii Plant Dis. 88 1318 1327

    • Search Google Scholar
    • Export Citation
  • Nishijima, K.A., Couey, H.M. & Alvarez, A.M. 1987 Internal yellowing, a bacterial disease of papaya fruits caused by Enterobacter cloacae Plant Dis. 71 1029 1034

    • Search Google Scholar
    • Export Citation
  • Nishijima, K.A., Wall, M.M. & Siderhurst, M.S. 2007a Demonstrating pathogenicity of Enterobacter cloacae on macadamia and identifying associated volatiles of gray kernel of macadamia in Hawaii Plant Dis. 91 1221 1228

    • Search Google Scholar
    • Export Citation
  • Nishijima, K.A., Keith, R.C., Fitch, M.M., Wall, M.M., Sugiyama, L.S. & Nishijima, W.T. 2007b Production of internal yellowing symptoms on resistant and susceptible papaya cultivars by Enterobacter cloacae at varying inoculum concentrations Phytopathology 97 S84

    • Search Google Scholar
    • Export Citation
  • Raybaudi-Massilia, R.M., Mosqueda-Melgar, J., Soliva-Fortuny, R. & Martin- Belloso, O. 2009 Control of pathogenic and spoilage microorganisms in fresh-cut fruits and fruit juices by traditional and alternative natural antimicrobials Comp. Rev. Food Sci. Food Safety 8 157 180

    • Search Google Scholar
    • Export Citation
  • Richard, C. 1984 Genus VI. Enterobacter Hormaeche and Edwards 1960, 72; Nom. Cons. Opin. 28. Jud. Comm. 1963, 38 465 469 Kreig N.R. & Holt J.G. Bergey's manual of systemic bacteriology Vol. 1 Williams & Wilkins Baltimore, MD

    • Search Google Scholar
    • Export Citation
  • Rosen, H.R. 1922 The bacterial pathogen of corn stalk rot Phytopathology 12 497 499

  • Ross, A.I.V., Griffiths, M.W., Mittal, G.S. & Deeth, H.C. 2003 Combining nonthermal technologies to control foodborne microorganisms Int. J. Food Microbiol. 89 125 138

    • Search Google Scholar
    • Export Citation
  • Sanders, W.E. & Sanders, C.C. 1997 Enterobacter spp.: Pathogens poised to flourish at the turn of the century Clin. Microbiol. Rev. 10 220 241

  • Schroeder, B.K., Waters, T.D. & du Toit, L.J. 2010 Evaluation of onion cultivars for resistance to Enterobacter cloacae in storage Plant Dis. 94 236 243

    • Search Google Scholar
    • Export Citation
  • Sela, S., Nestel, D., Pinto, R., Nemny-Lavyand, E. & Bar-Joseph, M. 2005 Mediterranean fruit fly as a potential vector of bacterial pathogens Appl. Environ. Microbiol. 71 4052 4056

    • Search Google Scholar
    • Export Citation
  • Sodexho Supplier Code of Practice 2007 Fruit & vegetables—Microbiological criteria: Prepared, ready-to-eat and frozen products 71 16 Aug. 2007 <http://uk.sodexo.com/uken/Images/Suppcodesmaller_tcm15-50823.pdf>.

    • Search Google Scholar
    • Export Citation
  • Takahashi, Y., Takahashi, K., Sato, M., Watanabe, K. & Kawano, T. 1997 Bacterial leaf rot of Odontioda orchids caused by Enterobacter cloacae Ann. Phytopathological Soc. Jpn. 63 164 169

    • Search Google Scholar
    • Export Citation
  • Wang, G.F., Praphat, K., Xie, G.L., Zhu, B. & Li, B. 2008 Bacterial wilt of mulberry (Morus alba) caused by Enterobacter cloacae in China Plant Dis. 92 483

    • Search Google Scholar
    • Export Citation
  • Wick, R.L., Rane, K.K. & Sutton, D.K. 1987 Mung bean sprout disease caused by Enterobacter cloacae Phytopathology 77 123 [abstr.].

  • Yonemura, R.K. 2009 Improved method of processing papayas for food safety and quality—IQF Dream, LLC 4 Aug. 2009 <http://www.reeis.usda.gov/web/crisprojectpages/206495.html>.

    • Search Google Scholar
    • Export Citation
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