Atypical Internal Yellowing of Papaya Fruit in Hawaii Caused by Enterobacter sakazakii.

Internal yellowing (IY) caused by Enterobacter cloacae and characterized by yellow discolored tissue surrounding the papaya (Carica papaya L.) seed cavity, diffuse margins, and the presence of a distinctly rotten odor was first reported in 1987 (3). Here we report the formation of atypical internal yellowing (AIY) in ripe papaya caused by the bacterium Enterobacter sakazakii. In surveys conducted from 2006 to 2007, 'Kapoho Solo' papayas grown in the Puna District of Hawaii Island were obtained from various packinghouses. After incubation at 27°C, the papayas were bisected and examined for symptoms of IY. Among papayas that were asymptomatic for IY, a dull, greenish yellow discoloration of the flesh with a distinct margin extending from the seed cavity into the pericarp was noted, along with a pungent odor. These symptoms occurred in 5 of the 500 fruit surveyed and bacterial populations were 102 to 103 CFU/g. Discolored tissue was aseptically excised, weighed, macerated, serially diluted in sterile distilled water (SDW), and plated onto modified peptone yeast extract medium (PT-M4) (4). The plates were incubated at 30°C for 24 to 48 h until single colonies were evident. After 48 h, colonies on PT-M4 were orange-red, convex and circular, and surrounded by a somewhat opaque 1-mm margin. After single colony purification, five strains were obtained. The strains, inoculated into oxidation/fermentation-glucose tubes and API 20E strips (bioMerieux, Inc., Durham, NC) incubated at 30°C, were shown to be facultative anaerobes and identified as E. sakazakii with a 98.4% certainty. Colonies plated onto tryptic soy agar (TSA) and incubated for 72 h at 25°C produced yellow pigmentation, indicative of E. sakazakii. Amplification by PCR with E. sakazakii-specific primers (2) yielded a 929-bp fragment, which was absent with E. cloacae and Pseudomonas aeruginosa template DNA. To confirm pathogenicity, cell suspensions at 109 CFU/ml of putative E. sakazakii strains RK07-05, RK07-06, and RK07-07 and E. cloacae (3) were inoculated by injection (0.5 ml per site) into one-third-ripe 'Kapoho Solo' papayas (six fruit per strain, inoculated at duplicate sites) and incubated at 27°C for 4 days. Control sites were injected with 0.5 ml of SDW. Fruit inoculation experiments were repeated. E. cloacae-inoculated sites produced typical IY as previously described (3), while the sites inoculated with the three E. sakazakii strains produced greenish yellow tissue (26% mean incidence), symptomatic of AIY. Control sites did not produce IY or AIY. Koch's postulates were fulfilled, and the identification of reisolated bacterial strains was confirmed with API 20E, PCR, and pigment production on TSA. Although less prevalent (1% incidence) than the typical IY produced by E. cloacae (3), E. sakazakii has the potential to affect quality and food safety of fresh and processed papaya products. E. sakazakii has been implicated in a severe form of neonatal meningitis, sepsis, and necrotizing enterocolitis (1). Research into the transmission and infection of papaya of this cross-domain pathogen merits further study. References: (1) D. H. Adamson. Clin. Microbiol. Newsl. 3:19, 1981. (2) A. Lehner et al. BMC Microbiol. 4:43, 2004. (3) K. A. Nishijima et al. Plant Dis. 71:1029, 1987. (4) K. A. Nishijima et al. Plant Dis. 88:1318, 2004.

Resin-purified digoxigenin-labeled DNA probes: an efficient protocol for mapping and fingerprinting sugarcane.

Nonradioactive Southern blotting using digoxigenin (DIG) has become routinely applied to the analysis of single-copy genes for genetic mapping because it is fast and safe. Previous studies indicate that DIG-labeled probes are suitable for single-gene detection in less complex genomes, but their efficient application to mapping a large octoploid genome has not been discussed. We developed a stream-lined procedure for nonradioactive restriction fragment length polymorphism mapping and DNA fingerprinting of sugarcane that combines DIG-11-dUTP and anion-exchange chromatography. In this report, we show that anion-exchange chromatography provides a reliable and simple technique for the resin-purification of large numbers of DIG-labeled DNA fragments 0.3-3.0 kb in size, and it is essential in minimizing contaminants and nonspecific signal.

Production of Haploid Saccharum spontaneum L. - Comparison of Media for Cold Incubation of Panicle Branches and for Float Culture of Anthers.

Means were sought to increase the frequency of haploid production from anther cultures of Saccharum spontaneum L. Excised preemerged panicle branches were incubated in modified Murashige-Skoog (MS), Nitsch (H), Gamborg (B5), and Chu (N6) media at 10 °C for 4 to 10 weeks prior to removal of the anthers for culturing at 27 °C. Anthers, either continuously cultured on liquid media or floated on liquid media 1 to 8 weeks prior to nurse culture, produced visible calli in 30 days. Nearly 1% of the anthers in one clone, SES 208, produced calli. The calli increased in mass on nurse medium containing 0.25 to 0.50 mg · l(-1) picloram as the auxin source and differentiated into green calli when the picloram was reduced to 0.05 to 0.125 mg · l(-1). The green calli developed into plants on lowered-picloram or picloram-free media. Calli and green plants were obtained from all three clones studied. Plants were regenerated from 100 out of 276 callus lines. Seventeen plant lines have been transferred to greenhouse culture. Four of seven plant lines checked for ploidy were haploid, two were diploid. A method utilizing stomate guard cell lengths was developed as a means to estimate ploidy level. The increased success in haploid callus and plant production is thought to be the result of long cold treatment of panicle branches, use of low salt media for panicle treatment in the cold, use of picloram in place of 2,4-D in the nurse culture, and use of a high nitrate/ammonium ratio in the liquid culture.

Somatic embryogenesis and plant regeneration from immature zygotic embryos of papaya (Carica papaya L.).

Immature zygotic embryos from open-pollinated and selfed Carica papaya L. fruits, 90 to 114 days post-anthesis, produced 2 to 20 somatic embryos on apical domes, cotyledonary nodes, and radicle meristems after culture for three weeks on half-strength Murashige and Skoog (MS) medium supplemented with 0.1 to 25 mg l(-1) 2,4-dichlorophenoxyacetic acid (2,4-D), 400 mg l(-1) glutamine, and 6% sucrose. After six weeks of culture, about 40 to 50% of the zygotic embryos had become embryogenic, and each embryogenic embryo yielded hundreds of somatic embryos within five months of culture on media supplemented with 2,4-D. Somatic embryos matured on half-strength MS medium, germinated on MS medium containing 5 mg l(-1) kinetin, and grew large enough for greenhouse culture on MS medium. Shoots were rooted in vermiculite and grown in the greenhouse.

Transgenic papaya plants from Agrobacterium-mediated transformation of somatic embryos.

Transgenic papaya (Carica papaya L.) plants were regenerated from embryogenic cultures that were cocultivated with a disarmed C58 strain of Agrobacterium tumefaciens containing one of the following binary cosmid vectors: pGA482GG or pGA482GG/cpPRV-4. The T-DNA region of both binary vectors includes the chimeric genes for neomycin phosphotransferase II (NPTII) and ß-glucuronidase (GUS). In addition, the plant expressible coat protein (cp) gene of papaya ringspot virus (PRV) is flanked by the NPTII and GUS genes in pGA482GG/cpPRV-4. Putative transformed embryogenic papaya tissues were obtained by selection on 150 µg·ml(-1) kanamycin. Four putative transgenic plant lines were obtained from the cp gene(-) vector and two from the cp gene(+) vector. GUS and NPTII expression were detected in leaves of all putative transformed plants tested, while PRV coat protein expression was detected in leaves of the PRV cp gene(+) plant. The transformed status of these papaya plants was analyzed using both polymerase chain reaction amplification and genomic blot hybridization of the NPTII and PRV cp genes. Integration of these genes into the papaya genome was demonstrated by genomic blot hybridizations. Thus, like numerous other dicotyledonous plant species, papayas can be transformed with A. tumefaciens and regenerated into phenotypically normal-appearing plants that express foreign genes.

Stable transformation of papaya via microprojectile bombardment.

Stable transformation of papaya (Carica papaya L.) has been achieved following DNA delivery via high velocity microprojectiles. Three types of embryogenic tissues, including immature zygotic embryos, freshly explanted hypocotyl sections, and somatic embryos derived from both, were bombarded with tungsten particles carrying chimeric NPTII and GUS genes. All tissue types were cultured prior to and following bombardment on half-strength MS medium supplemented with 10 mg 1(-1) 2,4-D, 400 mg 1(-1) glutamine, and 6% sucrose. Upon transfer to 2,4-D-free medium containing 150 mg 1(-1) kanamycin sulfate, ten putative transgenic isolates produced somatic embryos and five regenerated leafy shoots. Leafy shoots were produced six to nine months following bombardment. Tissues from 13 of these isolates were assayed for NPTII activity, and 10 were positive. Six out of 15 isolates assayed for GUS expression were positive. Three isolates were positive for both NPTII and GUS.

Molecular markers for sex determination in papaya ( Carica papaya L.).

We have developed molecular markers tightly linked to Sex1, the gene that determines plant sex in papaya ( Carica papaya L.). Three RAPD products have been cloned and a portion of their DNA sequenced. Based on these sequences SCAR primers were synthesized. SCAR T12 and SCAR W11 produce products in hermaphrodite and male plants and only rarely in females. SCAR T1 produces a product in all papayas regardless of plant sex. SCAR T12 and SCAR W11 showed no recombination in a population of 182 F2 plants from a 'SunUp' by 'Kapoho' cross. Based on these results a PCR-based technique for rapidly and accurately determining the sex of papaya plants was developed using either W11 or T12 to detect the hermaphrodite or male allele and T1, which amplifies a product regardless of sex type, as a positive control. The sexing technique, using SCAR T12 and SCAR T1 as a positive control, was used to correctly predict hermaphrodite papaya plants in a population of seedlings with an overall accuracy of 99.2%.

Genetic diversity of Carica papaya as revealed by AFLP markers.

Genetic relationships among Carica papaya cultivars, breeding lines, unimproved germplasm, and related species were established using amplified fragment length polymorphism (AFLP) markers. Seventy-one papaya accessions and related species were analyzed with nine EcoRI-MseI primer combinations. A total of 186 informative AFLP markers was generated and analyzed. Cluster analysis suggested limited genetic variation in papaya, with an average genetic similarity among 63 papaya accessions of 0.880. Genetic diversity among cultivars derived from the same or similar gene pools was smaller, such as Hawaiian Solo hermaphrodite cultivars and Australian dioecious cultivars with genetic similarity at 0.921 and 0.912, respectively. The results indicated that self-pollinated hermaphrodite cultivars were as variable as open-pollinated dioecious cultivars. Genetic diversity between C. papaya and six other Carica species was also evaluated. Carica papaya shared the least genetic similarity with these species, with an average genetic similarity of 0.432; the average genetic similarity among the six other species was 0.729. The results from AFLP markers provided detailed estimates of the genetic variation within and among papaya cultivars, and supported the notion that C. papaya diverged from the rest of Carica species early in the evolution of this genus.