Lisa M. Keith

Identification and characterization of Pestalotiopsis spp. causing scab disease of guava, Psidium guajava, in Hawaii

Guava is one of the most widely grown plants in the tropics; however, it is affected by many fruit rot diseases. Fruit diseases decrease the marketability of fresh fruit and fruit for processing. A survey of scab disease was conducted at the USDA/ARS Tropical Plant Genetic Resource Management Unit in Hilo, HI, where more than 50 accessions of guava are grown. Symptoms observed were gray/light brown lesions surrounded by dark brown borders on leaves and brown, raised, corky, necrotic lesions on the exocarp of fruit which progressed as the fruits matured. Seventeen isolates from infected fruit, six isolates from lesions on leaves, and nine isolates from additional crops surrounding the guava trees were collected. The main fungi consistently isolated from symptomatic leaves and fruit were Pestalotiopsis spp. Morphology, colony characteristics, and pathogenicity of the isolates were examined and potential sources of host resistance were identified for germplasm characterization studies. Molecular methods were used to identify four Pestalotiopsis taxa (P. clavispora, P. microspora, P. sp. GJ-1, and P. disseminata) on guava in Hawaii. To our knowledge, this is the first report of traditional and molecular methods of identification and characterization being used for fungal pathogens of guava in Hawaii.

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.

Biogas energy production from tropical biomass wastes by anaerobic digestion

Anaerobic digestion (AD) is an attractive technology in tropical regions for converting locally abundant biomass wastes into biogas which can be used to produce heat, electricity, and transportation fuels. However, investigations on AD of tropical forestry wastes, such as albizia biomass and food wastes, such as taro, papaya, and sweet potato, are limited. In this study, these tropical biomass wastes were evaluated for biogas production by liquid AD (L-AD) and/or solid-state AD (SS-AD), depending on feedstock characteristics. When albizia leaves and chips were used as feedstocks, L-AD had greater methane yields (161 and 113 L kg(-1)VS, respectively) than SS-AD (156.8 and 59.6 L kg(-1)VS, respectively), while SS-AD achieved 5-fold higher volumetric methane productivity than L-AD. Mono-digestion and co-digestion of taro skin, taro flesh, papaya, and sweet potato achieved methane yields from 345 to 411 L kg(-1)VS, indicating the robustness of AD technology.

Isolation and Characterization of Burkholderia gladioli from Orchids in Hawaii

Bacterial diseases of orchids continue to be serious problems. Bacterial strains were isolated from orchid plants exhibiting disease symptoms in Hawaii. Small to large leaf spots with or without water-soaking or soft rots were observed on various orchid genera, including Dendrobium, Oncidium, and Miltonia spp. and hybrids. Bacteria isolated and cultured from the lesions were tentatively identified using analytical profile index (API) strips and standard physiological and biochemical tests, and confirmed by species-specific polymerase chain reaction and sequencing of the 16S rRNA gene. The variation in pathogenic, morphological, cultural, and molecular characteristics of the orchid isolates also was evaluated. In our studies, a gramnegative, aerobic, rod-shaped bacterium that produced pale yellow, opaque, round colonies with entire margins on nutrient broth yeast extract agar (NBY) was isolated consistently from diseased orchid plants. On yeast dextrose calcium carbonate agar, the isolates produced brownishyellow, nonmucoid colonies, with the majority of the strains secreting a diffusible yellow or tan pigment into the media. The bacterium was identified as Burkholderia gladioli. Molecular analysis indicated very little diversity in the 16S rDNA gene. Testing B. gladioli isolates using media containing copper or streptomycin indicated varying levels of resistance (copper resistant = Cur; streptomycin resistant, Smr), with approximately 75% of the strains resistant to copper and 94% of the strains resistant to streptomycin. The minimum inhibitory concentration (MIC) of cupric sulfate among Cur strains ranged from 50 to 1,000 μg/ml and the MIC of streptomycin was 50 to 100 μg/ml for all Smr B. gladioli strains tested. Field and laboratory data suggest the frequent use of these chemicals in nurseries may have inadvertently resulted in the development of copper and streptomycin resistance in B. gladioli from orchids.

First Report of Ceratocystis Wilt on ˋŌhiˋa (Metrosideros polymorpha)

ˋŌhiˋa (Metrosideros polymorpha Gaudich.) is Hawaiˋi’s most common and widespread native tree, occurring from sea level to 2500-m elevation in both dry and wet forests and on substrates ranging from 50 to 4 million years in age (Friday and Herbert 2006). It is the most ecologically important native Hawaiˋian tree, defining native forest succession and ecosystem function over broad areas, providing critical habitat for rare and endangered native bird and insect species, and exemplifying the strong links between native Hawaiˋian culture and the islands’ environment (Dawson and Stemmermann 1990). Within the past 5 years, extensive ˋōhiˋa mortality has been observed in the Puna District of Hawai‘i Island in previously healthy trees growing in undisturbed forest settings. Affected trees exhibit rapid, synchronized death of leaves on individual branches that eventually spreads to the entire canopy. Dark-brown to black discoloration can be seen in the woody xylem of affected trees. Five branch samples from two trees displaying characteristic symptoms were collected in February and June 2014 from Leilani Estates subdivision (19°28′ N, 154°55′ W). A fungus that produced perithecia and formed olive-brown cultures on 10% V8 agar was consistently isolated from infected tissue. After 14 days, the perithecia were black, globose, 143 to 275 × 110 to 264 μm, and possessed a long, black neck (660 to 880 μm). Ascospores were hat-shaped, hyaline, and 5.7 to 8.6 × 2.7 to 4.3 μm. Hyaline, cylindrical endoconidia (14.3 to 38.6 × 2.9 to 4.3 μm) were found. PCR amplification and sequencing of the ITS region of rDNA were carried out for one isolate, P14-1-1. BLAST analysis of the sequence data (GenBank Accession No. KP203957) showed that the isolate was 98.9% homologous (525/531 nt) to Ceratocystis fimbriata (KC493164, derived from CBS115167). Strain KC493164 and others with 100% homology were collected from Syngonium spp. in Hawaii, Florida, Brazil, and Australia (Thorpe et al. 2005). Based on these morphological and molecular characteristics the fungus was identified as C. fimbriata (Engelbrecht and Harrington 2005). Pathogenicity was tested on 1to 2-year-old M. polymorpha seedlings as follows. Eight healthy plants were wounded with a scalpel ∼50 cm above the soil level. Two filterpaper disks soaked in a 10-spore/ml suspension was inserted in a stem flap and wrapped with parafilm. Control plants were inoculated with filter disks soaked in sterile water. The treated plants were incubated at 24°C with 12-h light in a humid chamber. Wilt symptoms were observed on 7 of 8 plants within 17 to 36 days. As the disease progressed, leaves withered, died, and remained attached to the plant. Plant death occurred around 100 days. Internal discoloration was observed in the main stem. C. fimbriata was successfully reisolated and identified morphologically from the infected seedlings. All control plants remained healthy. To our knowledge, this is the first report of C. fimbriata causing disease in M. polymorpha. This pathogen poses a serious threat to Hawai‘i’s flagship native tree species whose loss would be catastrophic for the diversity, structure, and function of Hawaiˋi’s remaining native forests and the services they provide.

Identification of Members of the Dimocarpus Longan Flowering Locus T Gene Family with Divergent Functions in Flowering

Dimocarpus longan is a subtropical fruit crop whose year-round production relies on the application of KClO3 to induce flowering; however, the mechanism by which this chemical causes flowering is yet unknown. To further characterize floral signaling in this species, we have isolated three longan FLOWERING LOCUS T (FT)-like genes and studied their activities by heterologous expression in Arabidopsis. Expression of two of these genes (DlFT2 and DlFT3) accelerates flowering, whereas expression of the third gene (DlFT1) causes delayed flowering and produced floral morphology defects. This anti-florigenic protein may be a member of a class of FT-like family involved in flowering time control in biennial and perennial species. Surprisingly, KClO3 treatment also suppressed the expression of both DlFT2 and DlFT3 in a field trial.

Organ-Specific Transcriptome Profiling of Metabolic and Pigment Biosynthesis Pathways in the Floral Ornamental Progenitor Species Anthurium amnicola Dressler

Anthurium amnicola Dressler possesses a number of desirable and novel ornamental traits such as a purple-colored upright spathe, profuse flowering, and floral scent, some of which have been introgressed into modern Anthurium cultivars. As a first step in identifying genes associated with these traits, the transcriptome from root, leaf, spathe, and spadix from an accession of A. amnicola was assembled, resulting in 28,019 putative transcripts representing 19,458 unigenes. Genes involved in pigmentation, including those for the metabolism of chlorophyll and the biosynthesis of carotenoids, phenylpropanoids, and flavonoids were identified. The expression levels of one MYB transcription factor was highly correlated with naringenin 3-dioxygenase (F3H) and dihydroflavonol-4-reductase (DFR) in leaves, whereas a bHLH transcription factor was highly correlated with flavonoid 3′-monooxygenase (F3′H) and a DFR in spathes, suggesting that these two transcription factors might regulate flavonoid and anthocyanin synthesis in A. amnicola. Gene sequence and expression data from four major organs of A. amnicola provide novel basal information for understanding the genetic bases of ornamental traits and the determinants and evolution of form and function in the Araceae.

A Spectral Mapping Signature for the Rapid Ohia Death (ROD) Pathogen in Hawaiian Forests

Pathogenic invasions are a major source of change in both agricultural and natural ecosystems. In forests, fungal pathogens can kill habitat-generating plant species such as canopy trees, but methods for remote detection, mapping and monitoring of such outbreaks are poorly developed. Two novel species of the fungal genus Ceratocystis have spread rapidly across humid and mesic forests of Hawaiʻi Island, causing widespread mortality of the keystone endemic canopy tree species, Metrosideros polymorpha (common name: ʻōhiʻa). The process, known as Rapid Ohia Death (ROD), causes browning of canopy leaves in weeks to months following infection by the pathogen. An operational mapping approach is needed to track the spread of the disease. We combined field studies of leaf spectroscopy with laboratory chemical studies and airborne remote sensing to develop a spectral signature for ROD. We found that close to 80% of ROD-infected plants undergo marked decreases in foliar concentrations of chlorophyll, water and non-structural carbohydrates, which collectively result in strong consistent changes in leaf spectral reflectance in the visible (400–700 nm) and shortwave-infrared (1300–2500 nm) wavelength regions. Leaf-level results were replicated at the canopy level using airborne laser-guided imaging spectroscopy, with quantitative spectral separability of normal green-leaf canopies from suspected ROD-infected brown-leaf canopies in the visible and shortwave-infrared spectrum. Our results provide the spectral–chemical basis for detection, mapping and monitoring of the spread of ROD in native Hawaiian forests.

First Report of Pseudomonas cichorii Causing Bacterial Leaf Blight of Plumeria pudica in Hawaii

Bridal Bouquet Plumeria (Plumeria pudica) is a commonly grown ornamental in Hawaii. In November 2014, a nursery grower located in Panaewa on the eastern side of Hawaii Island noticed water-soaked, irregular greyish-black leaf spots and leaf blight followed by defoliation on potted plants of P. pudica. Two leaf samples were submitted to the University of Hawaii Agricultural Diagnostic Service Center located at the Komohana Research and Extension Center in Hilo, HI for diagnosis. Isolations were made on King’s Medium B agar (KMB) and bacteria were consistently isolated from diseased tissue. Culture plates were forwarded to the USDA-ARS facility in Hilo, HI for further purification and molecular identification. Isolations on KMB resulted in essentially pure cultures that were non-mucoid, cream and fluorescent under 365 nm UV light. Four representative bacterial isolates were examined and characterized as gram-negative, oxidase positive, and produced a hypersensitive reaction in tobacco. Colonies were identif...