Ph.D. - Tropical Plant and Soil Sciences

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    EXPLORING PHYTOPHTHORA PALMIVORA PARTIAL RESISTANCE IN CARICA PAPAYA FOR CROP IMPROVEMENT
    (2024) Domingo, Ryan S.; Kantar, Michael; Tropical Plant and Soil Sciences
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    Breeding Scheme Development and Optimization During Neo-Domestication and Wide Hybridization
    (2023) Fumia, Nathan; Kantar, Michael B.; Tropical Plant and Soil Sciences
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    Anthurium Flower Color: Histology And Genetic Manipulation
    (University of Hawaii at Manoa, 2022) Toves, Peter J.; Amore, Teresita D.; Tropical Plant and Soil Sciences
    Anthurium is the most important cut flower in the Hawaii floriculture industry. Conventional breeding for desirable vase life, shape, and color has been the mainstay for keeping growers in Hawaii competitive in the global market. No blue anthuriums exist and cannot be made via conventional breeding since anthuriums lack the genes to produce delphinidin-derived anthocyanins. The objectives of this research were to expand our understanding of the biochemical and histological aspects of flower color that are essential for developing strategies for breeding, conduct transient gene expression in anthurium spathes, and to explore genetic engineering of the anthurium flavonoid pathway for novel color development. Flower color is influenced by pH, since anthocyanins exist in different forms depending on the pH of the vacuole. A survey of spathe pH in 50 hybrid selections and cultivars revealed that green spathes had the highest average pH (6.14), followed by whites (5.96), purples (5.82), pinks (5.78), corals (5.72), reds (5.46), and oranges (5.40). Flower color is influenced by the distribution and combinations of anthocyanins in the floral tissue. Anthocyanins were distributed in the hypodermis and mesophyll in red, pink, orange and coral anthurium spathes, with some hybrid selections having very few pigmented cells in the epidermal layer. The spatial distribution of anthocyanins was more expansive in purple anthurium spathes, and was observed in the adaxial and abaxial epidermis, and in the adaxial and abaxial hypodermis of some selections/cultivars. White and green spathes lacked anthocyanins; however green spathes contained substantial amounts of chlorophyll. The pH and spatial distribution data can help to make informed decisions when using the surveyed anthurium cultivars/selections for breeding, since pH can affect color and spatial distribution of anthocyanins can affect the perceived color intensity. Since the development of stable transformants with genes for novel flower color is a lengthy process with low transformation efficiency, we tested agroinfiltration-mediated transient expression to assess functionality of the structural gene F3’5’H and the transcription factors Delila and Rosea1 in 18 selections/cultivars. The effects of detached and attached spathes, older and younger spathes, the effect of full spectrum and enhanced LED, and sonication were tested in addition to agroinfiltration with the color constructs. The transient transformation results obtained were inconsistent and not conclusive for positive transient expression, since some spathes infiltrated without the color gene or transcription factors developed blue coloration despite the lack of F3’5’H or transcription factors Delila and Rosea1 . In this study, calli of seven hybrid selections or cultivars were co-cultivated with Agrobacterium tumefaciens AGL0 with one of the following plasmids with the NPTII selection gene: 1) pJAM1983 with the gene F3'5'H from petunia, 2) pJAM1889 with the gene Delila from Antirrhinum majus, 3) pJAM1463 harboring the gene Roseal from A. majus. Of the 372 samples tested via Polymerase Chain Reaction (PCR) (some made by Hawaii Agriculture Research Center (HARC), others made in this research), eight ‘Marian Seefurth’ samples from HARC tested positive for the NPTII antibiotic resistance gene and six of those samples tested positive for F3’5’H.
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    Shading Of Coffee Plantations For Induced Epizootics Of The Entomopathogenic Fungus Beauveria Bassiana (Bals.) Vuill. On The Coffee Berry Borer, Hypothenemus Hampei Ferrari
    (University of Hawaii at Manoa, 2020) Caraballo Ferrer, Jeffrey; Wright, Mark G.; Tropical Plant and Soil Sciences
    The use of shade has generated a debate on whether shade trees are beneficial to coffee culture. This topic has lacked a clear consensus in the scientific literature. In this research we addressed the effect of shade from a pest management perspective focusing on the impacts of shade on the entomopathogenic fungus Beauveria bassiana and the main insect pest, the coffee berry borer (Hypothenemus hampei (Ferrari), Coleoptera; Curculionidae, CBB) in the field and on green coffee quality following Specialty Coffee Association standard (SCA). The effect of irrigation, artificial and natural shade on the efficacy of B. bassiana, CBB infestation and damage to green coffee were evaluated. The irrigation treatments consisted of a high, low aerial irrigation and no irrigations regimes. The artificial and natural shade treatments consisted of 30% shade cloth, 50% shade cloth, full sun, shade under the canopy of a shade tree and shade beside the canopy of a shade tree. In addition, distance from the shade was used as a surrogate measure of shade in a separate experiment. The 5 distances or coffee tree positions were: 1, coffee trees right next to the shade tree trunk; 2, coffee trees located at the edge of the canopy of the shade tree; 3, coffee trees located halfway between the edge of the canopy and the farthest projected shade; 4, coffee trees located at the farthest projected shade and 5 located farther away where no shade was received. These distances were oriented according to the eastern and western cardinal directions from the shade tree. CBB mortality by B. bassiana, CBB field infestation, CBB parchment infestation, lost green bean yield, percentage of severely and slightly bored beans (in green coffee). In 2016, artificial and natural shade treatment significantly increased CBB mortality by B. bassiana, but not in 2015. Aerial irrigation did not affect CBB mortality by B. bassiana nor CBB field infestation. Artificial and natural shade did not affect the CBB field infestation in 2015, but in 2016 dense shade increased the CBB field infestation. The CBB parchment infestation was affected by artificial and natural shade where in 2015 the infestation decreased with increasing shade and in 2016 CBB parchment infestation increased with increasing shade. The shade cloth treatments and shade beside the canopy had less green coffee yield loss and percentage of severely bored beans than full sun and shade under the canopy. Distance from the shade did not affect CBB mortality by B. bassiana. Distance from the shade tree significantly affected CBB parchment infestation, green coffee yield loss and percent of severely bored beans where the coffee trees beside the canopy of the shade tree that received cardinal shade had the lowest infestation, yield loss and percentage of severely bored beans. Cardinal direction also had a significant impact on CBB parchment infestation, yield loss and percent of severely bored green beans but the actual difference between the eat and west was small. In summary, no adverse effect of shade nor irrigation on CBB mortality by B. bassiana were recorded. Although, shade is capable of increasing CBB mortality by B. bassiana, this effect was not consistent. In addition, shade beside shade trees and lighter shade percentages reduce CBB damage to coffee green coffee quality.
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    Morphological And Molecular Approaches To Disentangling The Taxonomy Of Plumeria Species (apocynaceae)
    (University of Hawaii at Manoa, 2019) Perez, B. Kauahi; Manshardt, Richard M.; Tropical Plant and Soil Sciences
    This dissertation investigated the taxonomy of Plumeria, a popular ornamental plant in the Apocynaceae. A brief introduction to the plant and its current taxonomy is provided in Chapter 1 along with a proposal for research to identify Plumeria spp. by morphological and molecular approaches. The overall goal of this research was to evaluate morphological and molecular characters that are useful for identifying Plumeria spp. so that we can delineate species boundaries, verify our taxonomic understanding of Plumeria, and begin to understand their evolutionary history. The use of qualitative and quantitative morphology to diagnose Plumeria spp. from the literature is difficult because of the multitude of descriptions given by various authors, even for the same species. Furthermore, the criteria for delineating currently recognized Plumeria spp. is unclear. Hence, in Chapter 2 the use of descriptive morphology is evaluated to determine its effectiveness at identifying Plumeria spp. Using iterative principal component analyses, it was found that a combination of descriptive vegetative characters was useful for identifying most Plumeria spp. However, other species could not be identified based solely on descriptive morphology, due to morphological variation of descriptors used. Instead, it would require the use of quantitative measurements and the use of other morphological characters, such as fruits and flowers, to properly diagnose these species. Chapter 3 explores molecular approach to delineating species and investigates the phylogenetic utility of five candidate loci. Some regions were able to identify operational taxonomic units as true species, but no single region could be used to identify all the putative species in our sampling. In fact, not all species could be recovered as distinct clusters even with a data set that combined four molecular regions. On the other hand, it did result in a well resolved phylogeny that agrees with prior findings, notably that most Plumeria are distinguishable by molecular means and that some Plumeria form a species complex comprised of morphologically variable members that share very similar molecular characters. Chapter 4 concludes with a synthesis of morphological and molecular findings and future directions in the realm of disentangling the taxonomy of Plumeria spp.
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    Molecular Analyses Of Papaya Viruses In Bangladesh: Detection, Characterization, And Distribution
    (University of Hawaii at Manoa, 2019) Hamim, Islam; Hu, John S.; Tropical Plant Pathology
    Aphid-transmitted PRSV is the greatest disease threat to commercial papaya production worldwide. Specific, ultrasensitive assays are important for the early detection of PRSV in the field. I developed a single-tube nested PCR (STNP) assay to address this need. Two nested PCR primer sets were designed to target the P3 gene of the virus. The concentrations and annealing temperatures of both primer pairs were optimized to avoid potential competition between primer sets during STNP. The assay was more sensitive than regular RT-PCR as determined by serial dilutions of cDNA and RNA templates and sample extracts from infected plants. RT-PCR and ELISA were capable of detecting PRSV 14 to 21 days post-inoculation, whereas STNP detected PRSV in plants 7d post-inoculation. This new STNP assay also detected PRSV from virus-infected asymptomatic plants. This system could assist epidemiological studies in the field and in quarantine protocols by enabling early detection of very low PRSV titers in the field and in imported plant samples. As in other countries, PRSV is the major limitation to papaya production in Bangladesh. Full-length coding genomes of PRSV strains from severely infected papaya plants were determined using the Illumina NextSeq 500 platform, followed by Sanger DNA sequencing of viral genomes obtained by reverse transcriptase polymerase chain reaction (RT-PCR). The genome sequences of two distinct PRSV strains, PRSV BD-1 (10,300 bp) and PRSV BD-2 (10,325 bp) were 74% and 83% identical to each other at the nucleotide and amino acid levels, respectively. PRSV BD-1 and PRSV BD-2 were 74 to 75% and 79 to 88% identical to other full-length PRSV sequences at the nucleotide level, respectively. Based on phylogenetic analysis, PRSV BD-2 was most closely related to PRSV-Meghalaya (MF356497) from papaya in India. PRSV BD-1 formed a distinct branch from the other PRSV sequences by nucleotide as well as amino acid sequence comparisons. Comparisons of the genome sequences of these newly identified PRSV strains with other sequenced PRSV genomes indicated two putative recombination events in PRSV BD-2. One recombinant event contained a 2,766-nucleotide fragment with the highest identity to PRSV-Meghalaya (MF356497) and the other contained a 5,105-nucleotide fragment with the highest identity to PRSV-China (KY933061). The occurrence of PRSV BD-1 and PRSV BD-2 in the sampled areas of Bangladesh was approximately 19% and 69%, respectively. Plants infected with both strains (11%) exhibited more severe symptoms than plants infected with either strain alone. The new full-length genome sequences of these PRSV strains from Bangladesh and their distribution provide important information on the dynamics of papaya ringspot virus infections in papaya in Bangladesh. Forty-five papaya samples with severe leaf curl symptoms were tested by PCR using a degenerate primer set for virus species in the genus Begomovirus. Of these, 29 were positive for tomato leaf curl Bangladesh virus (ToLCBV). The complete genome sequences of ToLCBV (GenBank accession no. MH380003) and its associated tomato leaf curl betasatellite (ToLCB) (MH397223) from papaya isolate Gaz17-Pap were determined and characterized. Defective betasatellites were found in ToLCBV-positive papaya isolates Gaz19-Pap, Gaz20-Pap and Gaz21-Pap. This study confirmed that papaya is a host of ToLCBV, ToLCB, and other defective and recombinant DNA satellites in Bangladesh. The complete genome sequence of tomato leaf curl Joydebpur virus (ToLCJoV) and its associated tomato leaf curl Joydebpur betasatellite (ToLCJoB) were determined and characterized from papaya isolate J1-Pap. ToLCJoV infecting papaya was most closely related to ToCJoV reported from Gazipur, Bangladesh, causing tomato leaf curl disease of tomato. ToLCB infecting papaya had the highest homologies at nucleotide and amino acid levels to ToLCB from tomato in India. The complete genome sequences of the DNA A and DNA B components of tomato leaf curl New Delhi virus (ToLCNDV) were determined and characterized from papaya isolate ND-71. ToLCNDV infecting papaya was most closely related to ToLCNDV reported from Gazipur, Bangladesh, causing tomato leaf curl disease of tomato. DNA B of ToLCNDV infecting papaya had the highest homologies at nucleotide and amino acid levels of DNA B of ToLCNDV from tomato in India.
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    Management Of Plant-parasitic Nematodes And Soil Health Using Oil Radish (Raphanus sativus) And Brown Mustard (Brassica juncea) Cover Crops
    (University of Hawaii at Manoa, 2019) Waisen, Philip; Wang, Koon-Hui; Tropical Plant Pathology
    This dissertation is composed of five chapters. Chapter one reviews factors that affect efficacy of biofumigation on management of plant-parasitic nematodes, focussing on Meloidogyne spp. in cropping systems and highlights two challenges. First, a number of literatures highlighted that susceptibility of biofumigant crops to target plant-parasitic nematodes could be an important management challenge and second, biofumigation being biocidal could have non-target impacts on free-living nematodes as bioindicators of soil health. Chapter two takes an alternative approach and elucidates the challenge of susceptibility as an opportunity to capitalize on as a trap crop arguing that using a good brassica host to a target nematode would be more effective as a conventional trap crop than using a poor host. When comparing trap cropping effects of ‘Sodbuster’ oil radish (OR; Raphanus sativus) as a poor host and ‘Caliente 199’ brown mustard (MS; Brassica juncea) as a good host against Meloidogyne spp. and R. Reniformis, MS showed potential as a trap crop depending on how long the trap crop was grown. MS suppressed soil population densities of Meloidogyne spp. in first and second trials by 60 and 50%, respectively where the cover crop was terminated within 42 days after planting (DAP; P ≤ 0.05) but not in third trial when terminated 49 DAP. However, population densities of R. reniformis were not suppressed by MS in the first two trials where it was terminated 42 DAP but were suppressed by 61% (P ≤ 0.05) in the third trial when the MS was terminated 49 DAP. Chapter three confirmed with previous studies that tissue maceration is necessary to activate myrosinase-glucosinolate system in brassica tissues to release bioactive isothiocyanates (ITC), soil tillage is required for the tissues to be in contact with the nematodes, and covering black plastic mulch is important to retain ITC from volatilization loss, together to maximize biofumigation effect on Meloidogyne spp. and R. reniformis. In three field trials conducted using OR and MS, soil populations of Meloidogyne spp. were suppressed by OR or MS if the biofumigant crops were macerated (M), tilled (T) into the soil and covered with black plastic (BP) in all the trials, and reduced zucchini root galls in Trials I and II. However, suppression of Meloidogyne spp. was stronger when using MS than OR in the MTBP treatment. Regardless, MTBP suppressed R. reniformis in Trial I but not in Trials II and III. None-the-less, the trend appeared that MTBP reduced R. reniformis by 33.9 and 54.9% in Trials II and III, respectively. MTBP also stimulated zucchini growth in Trials I and III, but not in II. Chapter four investigated whether biofumigation could have non-target impacts on free-living nematode as indicators of soil health. Both OR and MS did not compromise soil health but instead OR enhanced nutrient enrichment throughout zucchini growth while MS did transiently for up to 1 month after biofumigation. Terminating both OR and MS by MTBP enhanced soil health indicators but suppressive to plant-parasitic nematodes. As indicators of biofumigation, Myr activity (based on soil glucose analysis) and soil sulfate analysis were conducted to establish relationships with soil health indicators and other response variables. Myrosinase activity had a strong positive relationship with soil health indicators when toluene (methylbenzene) was added in soil samples to arrest microbial degradation of glucose. However, sulfate was stable in the soil without toluene and even had a stronger positive relationship with the soil health indicators, thus a good indicator of biofumigation in the field. Chapter five concludes the findings and provide recommendations and future directions to enhance biofumigation effects of brassicaceous cover crops against plant-parasitic nematodes.
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    Determining Potassium (K+) Release, Crop Availability and Uptake from Three Red Algal (Rhodophyta) Species
    (University of Hawaii at Manoa, 2017-05) Gangaiah, Chandrappa; Tropical Plant and Soil Sciences
    As a result of increasing fertilizer costs associated with rising oil prices, many growers in the Pacific region have become interested in locally available resources that can be used as low cost inputs to improve crop health and productivity. In recent years, use of algae and their extracts have gained in popularity due to their potential use in sustainable farming and may be used as an alternate to synthetic fertilizers. In Hawaii efforts are underway to control the most commonly found invasive algae species; Eucheuma denticulatum, Gracilaria salicornia, and Kappaphycus alvarezii. These are dominant invasive, non-indigenous species on Hawaiian reefs. The average potassium (K+) dry matter content in these species is around 14 -20%. Although these seaweeds were used in the past for crop production, not much is understood about application rates, yield performance, species efficacy for crop nutrition, nutrient release pattern and mechanisms of release. To address these gaps, greenhouse and laboratory experiments were conducted to: 1) determine the efficiency of the cardy meter as a rapid, low-cost tool for evaluating the tissue K status of pak choi from fresh sap, 2) describe effect of three invasive algae species on growth (yield) and K nutrition (tissue K+) of pak choi at different K fertilizer rates, 3) to evaluate algae biomass as a replacement for K+ synthetic fertilizers, 4) compare the two buffers to minimize ionic interference using a K+ selective electrode, and 5) to understand the K+ nutrient release pattern and mechanics of these three species. Our data show that the invasive algae species increased the yield and growth of pak choi and the response was greater when algae was applied to provide K+ at the rate of 224-284 kg.ha-1. Results of the algae comparison with synthetic K+ showed no significant differences between them for yield; e.g. Plants grown with K. alvarezii, KCl and KNO3 had an average dry weight of 7.5 g when K+ was provided at 280 kg.ha-1. Only K+ rates, not K+ source were significant for yields and tissue K concentrations in all trials. Although the values between rapid electrode and ICP measured tissue K+ values were well correlated, ICP was identified as the preferred method of K+ quantification in tissue. However, in soil solution, ion strength adjustment buffers (ISAB) were able to significantly mask the other ionic interferences when measuring K+ and with a selective electrode. No notable difference was observed among the two buffers, NaClO4 and NaCl (slope near 1) with a strong correlation (r =0.99). This is the first report of such a comparison in the literature. Based on the results from the leachate study, we conclude that the total amount of K+ released from algae was lower than synthetic K fertilizers applied at equivalent rates of K+. Results from the both the whole plant and polysaccharides leachate studies show that there is a difference in K release pattern among the algae with the cumulative recovery of K+ from both whole plant G. salicornia and extracted agar were significantly lower (10-12%) than whole plant K. alvarezii and extracted carrageenan at both K application rates. These differences in release among algal species may be explained in part by differences in cell wall composition, chemical properties including sulfate groups differences and physical properties such as higher gel strength of Gracilaria agar. To our knowledge, this work is the first detailed report of these red algae species as significant sources of K+. This is also the first report in the literature of the less expensive NaCl buffer being an effective substitute for NaClO4, allowing for cost effective, high-throughput analysis of soil solution K+. We propose that carrageenan containing species may be more efficient sources of K+ than those containing agar, but future studies are needed to elucidate the mechanisms for these differences. Our study may also provide the established protocols for further investigation of the functional properties of naturally extracted agar and carrageenan from these species and at different locations.