First Report of Association of Mucor circinelloides on Noni (Morinda citrifolia) in Hawaii.

Noni (Morinda citrifolia) is a popular medicinal plant found in tropical or subtropical regions of the world. The fruit and juice extracts have properties that are reportedly therapeutic for diabetes, high blood pressure, and certain types of cancer (1,4). In our studies on noni juice produced from fruit collected from the Kohala and Puna districts of the island of Hawaii from 2008 to 2010, Mucor circinelloides f. sp. circinelloides was isolated from 85% of 157 juice samples and observed with up to 75% incidence on fruit surfaces during fermentation processing in glass jars. Fungal growth, appearing 14 to 21 days in storage at 22°C, was pale yellow to tan brown and was associated with wounded surfaces. Single-spore strains, KN 06-2 (2006; ripe fruit puree) and KN 08-08 (2008; fermented juice; CBS 124110), identified by Centraalbureau voor Schimmelcultures by molecular methods were 97.3% similar in internal transcribed spacer sequence to the type strain (CBS 195.68). M. circinelloides f. sp. circinelloides strains (KN 08-08, KN 09-06, or KN 10-02) (2008 to 2010; fermented juice) were inoculated by pipetting an aliquot of 100 µl of fungus strain spore suspension (1 × 105 to 1.33 × 106 spores/ml) onto firm, yellow maturity noni fruit that were washed, surface disinfected, and either wounded (surface cuts) or nonwounded. Controls consisted of no inoculation and sterile distilled water (SDW) inoculation treatments. Ten to twenty each of wounded and nonwounded fruit comprised each inoculation treatment. Fruit were incubated in acrylic bins with a layer of distilled water at the bottom, and sealed with snap-on lids. The bins were incubated on a lab bench at 22 to 23°C under fluorescent lights. Fruits were evaluated for presence of fungal growth and severity of symptoms. To determine viability of spores on inoculated fruit without symptoms, surfaces were swabbed with sterile cotton swabs dipped in SDW, streaked on potato dextrose agar (PDA) plates, and incubated at 22°C under fluorescent lights. The inoculation experiment was conducted twice. Nonwounded fruit inoculated with M. circinelloides f. sp. circinelloides strains did not result in infections (KN 09-06 and KN 10-02) or produced slight mycelial growth (0 to 20%; KN 08-08). Wounded fruit inoculated with any of the three strains resulted in 85 to 100% infection of moderate severity. There were no infections in noninoculated or SDW treatments of nonwounded or wounded fruit. Koch's postulates were fulfilled with the reisolation of M. circinelloides f. sp. circinelloides from selected fruit exhibiting soft tissue, discoloration, and sporulating yellowish green mycelial growth. Swab washes from asymptomatic surfaces of inoculated nonwounded fruit resulted in the growth of M. circinelloides f. sp. circinelloides on PDA, proving viability of the spores and confirmed that the fungus is primarily pathogenic only on wounded fruit surfaces. To our knowledge, this is the first report of M. circinelloides as a wound pathogen of noni fruit. The quality of fermented noni juice may be affected by the presence of M. circinelloides f. sp. circinelloides but can be remedied by pasteurization that does not affect antitumor properties (unpublished data). This fungus is also a reported pathogen of mango (2) and peach (3). References: (1) J. Li et al. Oncol. Rep. 20:1505, 2008. (2) K. Pernezny and G. W. Simone. Phytopathol. News 34:25, 2000. (3) C. Restuccia et al. J. Food Prot. 69:2465, 2006. (4) M. Y. Wang et al. Acta Pharmacol. Sin. 23:1127, 2002.

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.

Demonstrating Pathogenicity of Enterobacter cloacae on Macadamia and Identifying Associated Volatiles of Gray Kernel of Macadamia in Hawaii.

Gray kernel is an important disease of macadamia (Macadamia integrifolia) that affects the quality of kernels, causing gray discoloration and a permeating, foul odor. Gray kernel symptoms were produced in raw, in-shell kernels of three cultivars of macadamia that were inoculated with strains of Enterobacter cloacae. Koch's postulates were fulfilled for three strains, demonstrating that E. cloacae is a causal agent of gray kernel. An inoculation protocol was developed to consistently reproduce gray kernel symptoms. Among the E. cloacae strains studied, macadamia strain LK 0802-3 and ginger strain B193-3 produced the highest incidences of disease (65 and 40%, respectively). The other macadamia strain, KN 04-2, produced gray kernel in 21.7% of inoculated nuts. Control treatments had 1.7% gray kernel symptoms. Some abiotic and biotic factors that affected incidence of gray kernel in inoculated kernels were identified. Volatiles of gray and nongray kernel samples also were analyzed. Ethanol and acetic acid were present in nongray and gray kernel samples, whereas volatiles from gray kernel samples included the additional compounds, 3-hydroxy-2-butanone (acetoin), 2,3-butanediol, phenol, and 2-methoxyphenol (guaiacol). This is believed to be the first report of the identification of volatile compounds associated with gray kernel.

Association of Enterobacter cloacae with Rhizome Rot of Edible Ginger in Hawaii.

Edible ginger is a popular spice crop that is grown in Hawaii primarily for the fresh market, and as such, rhizome quality is of paramount importance. In our studies, a Gram-negative, facultative anaerobic, rod-shaped bacterium was consistently isolated from decayed as well as symptomless ginger rhizomes. The bacterium was identified as Enterobacter cloacae by biochemical assays and 16S rDNA sequence analysis. Rot symptoms, which usually occurred in the central cylinder of the rhizome, were characterized by yellowish-brown to brown discolored tissue and firm to spongy texture. In inoculation experiments, ginger strains of E. cloacae produced basal stem and root rot, with foliar chlorosis and necrosis in tissue-cultured ginger plantlets, and discolored and spongy tissue in mature ginger rhizome slices and whole segments. In other hosts, ginger strains of E. cloacae caused internal yellowing of ripe papaya fruit and internal rot of onion bulbs. All strains that caused symptoms in inoculated plants were reisolated and identified as E. cloacae. Our studies suggest that E. cloacae can exist as an endophyte of ginger rhizomes, and under conditions that are favorable for bacterial growth, or host susceptibility, including maturity of tissues, rhizome rot may occur. Rhizome quality may be impacted by the presence of E. cloacae under conditions such as high temperature, high relative humidity, and low oxygen atmosphere that may affect the development of decay, and such conditions should be avoided during post-harvest handling and storage. The association of E. cloacae with a rhizome rot of ginger is a new finding.

First Report of Lasmenia sp. and Two Species of Gliocephalotrichum on Rambutan in Hawaii.

Rambutan (Nephelium lappaceum L.) is a tropical fruit grown in Hawaii for the exotic fruit market. Fruit rot was observed periodically during 1998 and 1999 from two islands, Hawaii and Kauai, and severe fruit rot was observed during 2000 in orchards in Kurtistown and Papaikou on Hawaii. Symptoms were characterized by brown-to-black, water-soaked lesions on the fruit surface that progressed to blackening and drying of the pericarp, which often split and exposed the aril (flesh). In certain cultivars, immature, small green fruits were totally mummified. Rambutan trees with high incidence of fruit rot also showed symptoms of branch dieback and leaf spot. Lasmenia sp. Speg. sensu Sutton, identified by Centraalbureau voor Schimmelcultures (Baarn, the Netherlands), was isolated from infected fruit and necrotic leaves. Also associated with some of the fruit rot and dieback symptoms were Gliocephalotrichum simplex (J.A. Meyer) B. Wiley & E. Simmons, and G. bulbilium J.J. Ellis & Hesseltine. G. simplex was isolated from infected fruit, and G. bulbilium was isolated from discolored vascular tissues and infected fruit. Identification of species of Gliocephalotrichum was based on characteristics of conidiophores, sterile hairs, and chlamydospores (1,4). Culture characteristics were distinctive on potato dextrose agar (PDA), where the mycelium of G. bulbilium was light orange (peach) without reverse color, while G. simplex was golden-brown to grayish-yellow with dark brown reverse color. Both species produced a fruity odor after 6 days on PDA. In pathogenicity tests, healthy, washed rambutan fruits were wounded, inoculated with 30 µl of sterile distilled water (SDW) or a fungus spore suspension (105 to 106 spores per ml), and incubated in humidity chambers at room temperature (22°C) under continuous fluorescent light. Lasmenia sp. (strain KN-F99-1), G. simplex (strain KN-F2000-1), and G. bulbilium (strains KN-F2001-1 and KN-F2001-2) produced fruit rot symptoms on inoculated fruit and were reisolated from fruit with typical symptoms, fulfilling Koch's postulates. Controls (inoculated with SDW) had lower incidence or developed less severe symptoms than the fungus treatments. Inoculation tests were conducted at least twice. To our knowledge, this is the first report of Lasmenia sp. in Hawaii and the first report of the genus Gliocephalotrichum on rambutan in Hawaii. These pathogens are potentially economically important to rambutan in Hawaii. G. bulbilium has been reported previously on decaying wood of guava (Psidium guajava L.) in Hawaii (2), and the fungus causes field and postharvest rots of rambutan fruit in Thailand (3). References: (1) J. J. Ellis and C. W. Hesseltine. Bull. Torrey Bot. Club 89:21, 1962. (2) D. F. Farr et al. Fungi on Plants and Plant Products in the United States. The American Phytopathological Society, St. Paul, MN, 1989. (3) N. Visarathanonth and L. L. Ilag. Pages 51-57 in: Rambutan: Fruit Development, Postharvest Physiology and Marketing in ASEAN. ASEAN Food Handling Bureau, Kuala Lumpur, Malaysia, 1987. (4) B. J. Wiley and E. G. Simmons. Mycologia 63:575, 1971.