Ph.D. - Tropical Plant Pathology

Permanent URI for this collectionhttps://hdl.handle.net/10125/20045

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Molecular and biological characterization of emerging plant viruses in Hawaii
(University of Hawai'i at Manoa, 2025) Wang, Xupeng; Melzer, Michael J.; Tropical Plant Pathology
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HARNESSING THE POWER OF SORGHUM-SUDANGRASS HYBRIDS TO TRANSFORM SOIL HEALTH IN ANNUAL ROW CROP SYSTEMS: A NARRATIVE FROM NEMATODES AND BEYOND.
(University of Hawai'i at Manoa, 2024) Paudel, Roshan; Wang, Koon-Hui; Tropical Plant Pathology
Sorghum and sorghum-sudangrass hybrids (SSgH, Sorghum bicolor) are fast-growing, carbon-rich, drought-tolerant cover crops known for their soil building capability and biofumigation property. However, practices to terminate the cover crops to maximize soil health enhancement effects often compromise the biofumigation potential of the cover crop. The overall goal of this dissertation was to explore approaches to maximize SSgH ability to improve soil health while suppressing soilborne pathogens (Meloidogyne incognita, Rotylenchulus reniformis, Fusarium commune). It was hypothesized that SSgH performance would vary by SSgH variety, and their soil health improvement ability would also differ by initial soil health conditions. This dissertation focused on the use of nematode community analysis and soil microbial profiles estimated by phospholipid fatty acid (PLFA) analysis as soil health indicators to understand the changes in soil food web structure over multiple cropping cycles of a SSgH and eggplant (Solanum melongena) rotation. This dissertation is organized into five chapters. Chapter one provides a comprehensive literature review on the use of SSgH cover cropping and the nematode community as indicators of soil health. Chapter one also addresses the challenge of maximizing the soil health benefits of SSgH while ensuring that their biofumigation capabilities are not compromised. Chapter two documents the first attempt to terminate several varieties of SSgH cover crops in a no-till system. Practicing SSgH cover cropping in a no-till system only improved soil moisture and increased the abundance of omnivorous nematodes (indicating improvement in the stability of the nematode communities) without suppressing plant-parasitic nematodes (PPNs) after an eggplant crop. This result was not surprising as it is anticipated that biofumigant from SSgH (hydrolysis of dhurrin to hydrogen cyanide, HCN) is highly volatile. Thus, it is important to incorporate SSgH residues into the soil. Chapter three adopts an alternative approach to evaluate the performance of SSgH cover crops in strip- and low-till systems. Strip-till method minimizes soil disturbance while capturing some of the HCN in the soil. To examine SSgH cover crops in two soil health conditions, a total of 4 SSgH-eggplant field trials were conducted at the Poamoho Experiment Station, University of Hawaii. Two of these trials were conducted successionally at Location 1 previously fallowed for more than 5 years (nutrient depleted and disturbed), whereas the other two were conducted successionally at a 5-year no-till cover crop site (Location 2, nutrient enriched but disturbed). In all field trials, 7 SSgH varieties were grown for 3 months, terminated using a flail mower followed by handheld tilling of 20-cm wide, 10-cm deep strips. Two successional trials were conducted at each location. As hypothesized, the performance of SSgH in a low-till system was found to be location-specific. A rapid response of nematode communities was observed in nutrient-depleted and disturbed soil (Location 1), but a more subtle or gradual response to SSgH was found in the already biologically active soil (Location 2). Nonetheless, low-till SSgH cover cropping improved soil health and managed plant-parasitic nematodes in both locations. This field study also demonstrated that comprehensive soil health improvements required more than one cropping cycle of SSgH-cash crop rotation to achieve top-down regulation of plant-parasitic nematodes. Once the soil became more enriched and structured, a multivariate analysis showed that eggplant yield was negatively related to the abundance of root-knot nematodes and root gall index on eggplant, but positively related to volumetric soil moisture, volumetric aggregate stability, water infiltration, SSgH biomass, enrichment index, structure index, and Solvita microbial respiration. These results supported the hypothesis that promoting soil health by SSgH strip-till cover cropping would lead to the suppression of plant-parasitic nematodes. Chapter four examines factors affecting the SSgH biofumigation effect, including SSgH varieties, age of SSgH, environmental stress, and mulching vs soil incorporation. Initially, dhurrin concentration from 2-month-old tissues of 7 varieties of SSgH was analyzed using high-performance liquid chromatography (HPLC) and was determined to be highest in ‘NX-D-61’ and ‘Latte’. This HPLC assay confirmed differential concentrations of dhurrin among SSgH varieties. Regression analysis between dhurrin content and the number of R. reniformis infecting cowpea roots revealed a strong negative relationship (r² = 0.69; p = 0.02). Greenhouse and field trials were then conducted to examine the age effect of SSgH on biofumigation against Meloidogyne incognita and Rotylenchulus reniformis. The shoot biomass of several SSgH varieties was harvested at 1, 2, and 3 months of growth and used as soil amendments compared to an unamended control. Two greenhouse bioassays showed that ‘NX-D-61’ and ‘Latte’ amendments were most suppressive against the female development of M. incognita on mustard green (Brassica juncea) regardless of SSgH age, although other varieties showed a decrease in biofumigation effect against M. incognita as SSgH matured. When tested against R. reniformis, tissue amendment of all varieties of SSgH suppressed R. reniformis development in cowpea (Vigna unguiculata) roots However, discrepancies were observed in the age effect of SSgH against R. reniformis between trials. In Trial I, 2-month-old amendments had the greatest suppression, whereas in Trial II, 1-month-old amendments were more suppressive to R. reniformis. This led to a notion of the environmental effect on the dhurrin content. The 2-month-old tissues of SSgH in Trial I were collected from the driest month, making 2-month-old tissues more potent than the 1-month-old tissues, consistent with the literature that suggested dryer conditions led to higher dhurrin content in SSgH. To further examine the biofumigation effect of SSgH on soil-borne fungal pathogens, 2-month-old ‘NX-D-61’ tissue was amended into or mulched on field soil infested with Fusarium commune and planted with mustard green in greenhouse pot trials. Soil incorporation was more effective than surface mulching of ‘NX-D-61’ in reducing the disease severity of F. commune on mustard green in two greenhouse trials. The effectiveness of ‘NX-D-61’ biofumigation in the field against F. commune was examined in two trials, but only one of these trials suppressed F. commune where the cover crop was terminated at 1-month old and following two successive plantings of SSgH-mustard green rotation. Chapter five presents the conclusion of the dissertation. Improvements in soil health through SSgH cover cropping depend on both the SSgH varieties and the initial soil health conditions. High-dhurrin SSgH varieties can generate effective biofumigants when soil-incorporated at 1- or 2-month old against soil-borne pathogens, M. incognita, R. reniformis, and F. commune, but the effect might be better if continuous strip- and low-till practice of SSgH-crop rotation for at least 2 times in the same location. This dissertation clarified factors that affect SSgH biofumigation against soil-borne pathogens, especially plant-parasitic nematodes, and demonstrated that the benefits of SSgH do not have to compromise each other. In fact, better soil health improvement by SSgH could lead to better suppression of plant-parasitic nematodes beyond that achieved by SSgH biofumigation alone.
GENOMIC ANALYSES AND PATHOGENICITY DETERMINANTS OF XANTHOMONADS ASSOCIATED WITH ARACEAE AND ARALIACEAE
(University of Hawai'i at Manoa, 2023) Chuang, Shu-Cheng; Arif, Mohammad; Tropical Plant Pathology
Araceae and Araliaceae, two significant plant families, encompass economically important ornamentals, including Anthurium, Dieffenbachia, Philodendron, and Syngonium (Araceae) and Hedera, Schefflera, Fatsia, and Polyscias (Araliaceae). These plants face threats from two bacterial pathogens, Xanthomonas axonopodis pv. dieffenbachiae (Xad) and X. hortorum pv. hederae (Xhh), causing leaf blight and leaf spot diseases globally for several decades. In previous studies, both Xad and Xhh strains were identified as highly heterogeneous groups and were subsequently reclassified into different Xanthomonas species. The Pacific Bacterial Collection at UHM houses over 100 Xad strains and about 20 Xhh strains isolated from various genera of Araceae and Araliaceae, respectively. The taxonomic positions of Xad and Xhh in our culture collection, along with their pathogenicity and virulence factors interacting with Araceae and Araliaceae, await further investigation. This dissertation aims to achieve two primary objectives: (i) To investigate the phylogenetic and taxonomic relationships of Xanthomonas spp. associated with Araceae and Araliaceae; (ii) To explore the molecular mechanisms of pathogenicity and virulence in Xanthomonas spp. associated with Araceae and Araliaceae. In the first study (Chapter 2), we focus on elucidating the phylogenetic and genealogical relationships among Xanthomonas species isolated from Araceae. Multi-locus sequence analysis with five housekeeping genes is employed, analyzing 59 representative strains from various Araceae hosts, temporal and geographical origins, and 100 closely related Xanthomonas and Stenotrophomonas species/pathovars. The results reveal diverse clustering, raising some certainties and some uncertainties about the taxonomic positions of Xad strains. This prompts an exploration of whole genome constituents for genes and gene clusters involved in pathogenicity in the following Chapter 3. In the second study (Chapter 3), we aim to determine the taxonomic positions of selected representative Xad strains and investigate potential pathogenicity factors, including the type III secretion system (T3SS) and type III secretion effectors (T3Es). Genome-scale data unveils accurate taxonomic positions, and the presence or absence of T3SS and T3Es suggests some Araceae strains as potential pathogens, while others are considered commensal strains. In Chapter 4, three new species, X. hawaiiensis sp. nov., S. aracearum sp. nov., and S. oahuensis sp. nov., associated with Araceae, are proposed based on the overall genomic relatedness indices. Pan- and core-genome analyses are conducted for new species along with other type strains of Xanthomonas and Stenotrophomonas species. Lastly, Chapter 5 comprehensively discusses Xhh strains isolated from P. guilfoylei. Based on phylogenetic analysis and genomic relatedness, these representative strains are reclassified as X. euvesicatoria. Comparative genomic analyses reveal that avirulent strain has a big deletion in the flagellar biosynthesis gene cluster 2, resulting in nonmotile and aflagellation. Validation of gene function through reverse mutagenesis and pathogenicity test on P. guilfoylei suggests that two flagellar component genes, fliR and flhB, might be virulence determinants in X. euvesicatoria- P. guilfoylei pathosystem.
Characterization Of The Virome In Pineapple (Ananas sp.) And Flowering Ginger (Alpinia purpurata) In Hawaii
(University of Hawaii at Manoa, 2022) Larrea-Sarmiento, Adriana Estefania; Hu, John S.; Tropical Plant Pathology
Pineapple (Ananas comosus) and flowering ginger (Alpinia purpurata) are two of Hawaii’s most economic and culturally valuable crops. Plant viruses that affect food and horticultural crops are important threats to Hawaii’s economy. They can have a significant negative impact on their host by reducing both decorative value and quality of propagated material and by causing great economic damage. Because of the relevance of new viruses with a potential threat to Hawaii’s agriculture, characterization of their host range, genetic diversity, and tri-trophic virus-host-vector relationships are essential to evaluating strategies for disease management. Pineapple is a pantropic crop whose production has been severely affected by a viral disease, mealybug wilt of pineapple (MWP). MWP is associated with members of the genus Ampelovirus known as the pineapple mealybug wilt-associated virus (PMWaV) complex. PMWaVs are spread by several mealybug species of Dysmicoccus sp. Pineapple mealybug wilt-associated virus 2 (PMWaV-2) is associated with MWP in Hawaii, Cuba, and Brazil, but not in Australia or China. This may suggest that there might be uncharacterized virus or viruses infecting pineapple and associated with the disease. Here, we report the use of RNA-sequencing technologies to characterize five new virus species in pineapple belonging to two families. Four (+) ssRNA viruses, with bipartite genome and each RNA segment encoding one large open reading frame (ORF), were clustered as members within the subgenus Cholivirus, genus Sadwavirus (family Secoviridae). The four viruses were characterized infecting commercial and germplasm pineapple accessions, and a public domain of the Transcriptome Shotgun Assembly (TSA) in the GenBank database. They belong to the pineapple secovirus (PSV) complex including PSV-A, PSV-B, PSV-C, and PSV-D. Additionally, another member in the PMWaV complex, PMWaV-6, was characterized from MWP-symptomatic field samples, and was also found infecting germplasm accessions. Being closely related to PMWaV-2, PMWaV-6 was clustered as a member in the genus Ampelovirus, subgroup I (family Closteroviridae). RNA-seq was also used to study the occurrence of viral populations associated with pineapple germplasm accessions maintained in the USDA-ARS NPGR at PBARC in Hilo, Hawaii. We discovered 69 viral sequences representing 10 members within the Ampelovirus, Sadwavirus, and Badnavirus genera. Genetic diversity and recombination events were found in members of the PMWaV complex, as were PSVs. PMWaV-1, -3, and -6 presented recombination events across the quintuple gene block, while no recombination events were found for PMWaV-2. The high recombination frequency of the RNA1 and RNA2 molecules from PSV-A and PSV-B were congruent with the diversity found by phylogenetic analyses. Here, we also report the development and improvement of RT-PCR diagnostic protocols for specific identification and detection of viruses infecting pineapple. The RT-PCR assays were designed based on the diverse viral populations characterized in this study. Given the high occurrence of recombination events and diversity found in these viruses in the Ananas germplasm, the reported and validated RT-PCR assays bring an important advance in the surveillance of viral infections of pineapple. Finally, the pineapple mealybug, Dysmicoccus spp., and the pineapple red mite, Dolichotetranychus floridanus, were identified as potential vectors of the ampelovirus PMWaV-6, and the two secoviruses PSV-A and PSV-B, respectively in our preliminary studies. A. purpurata is a perennial plant in the family Zingiberaceae with origins in the South Pacific Islands. As an ornamental and cut flower crop, flowering ginger generates an impact on the Hawaiian floriculture and plant nurseries which in 2020 reported sales estimated at around $81 million (356,000 for cut ginger stems). During the last decade, virus-like symptoms observed in A. purpurata in Hawaii were reported to be caused by banana bract mosaic virus (BBrMV, genus Potyvirus) and Canna yellow mottle virus (CaYMV, genus badnavirus). Symptoms caused by a single infection with BBrMV include chlorotic yellow streaks on leaves and stems and red-brown spindle-shaped streaks on the bracts. Although single and mixed infectious have been observed for CaYMV, the virus has not been clearly associated with specific viral-like symptoms. In 2018, a second badnavirus, banana streak GF virus (BSGFV) was identified infecting flowering ginger on Oahu, Hawaii. In the last five years, flowering ginger production in Hawaii has been declining. Stakeholders have reported a decline in crop vigor and yield affecting the production of flowering ginger and associated to outbreaks of a severe dieback syndrome. To study the association of viral infection with the slow decline syndrome observed in flowering ginger in Hawaii, we used RNA-seq and bioinformatic analyses, and virus indexing on samples collected from four islands. Viral sequences corresponding to six viruses were recovered from samples with virus-like symptoms. Three of these viruses, CaYMV (genus Badnavirus), and two novel viruses, Alpinia vein clearing virus (ApVCV, genus Ampelovirus) and Alpinia vein streaking virus (ApVSV; genus betanucleorhabdovirus), were associated with the slow decline syndrome. The other three viruses included the badnavirus, BSGFV and two potyviruses, BBrMV and the newly reported bean common mosaic virus (BCMV), were found in low incidence, and were thus not associated with the slow decline symptoms. Virus detection from potential vectors in tandem with transmission assays identified the mealybug Planococcus citri as vector of CaYMV and ApVCV, while the aphid Pentalonia caladii was identified as a vector of the novel ApVSV. Both P. citri and P. caladii are common pests of flowering ginger in Hawaii. Transmission of ApVSV was achieved using aphid colonies naturally feeding on ApVSV-infected flowering ginger plants and detached leaves experiments although transmission studies using aphids reared on an alternate host were not successful. This may suggest that host switch of the vector P. caladii may have a negative influence on the transmission of ApVSV. Results reported in this dissertation provide an insight into the association of viral infections with the slow decline syndrome observed in flowering ginger in Hawaii. The discovery and characterization of new viruses contribute to our knowledge of the diversity and characteristics of the virome of many crops. Our multiple-approach methodology using RNA-seq and PCR-based diagnostics were useful to characterize the virome present in two economically important crops in Hawaii. Virus characterization and development of robust detection methods, most often PCR-based, were undertaken simultaneously and offered a fundamental screening tool to index propagative material and field collected samples of flowering ginger and pineapple. The diagnostic PCR-based methods implemented in this study will be a foundational indexing resource for the obtention of virus-free plants. Such “clean plants” are the cornerstone of plant production for all propagated crops, including pineapple and flowering ginger.
Genomic Biology And Interactions Of Pectinolytic Bacteria
(University of Hawaii at Manoa, 2021) Boluk, Gamze; Arif, Mohammad; Tropical Plant Pathology
The soft rot Pectobacteriaceae, the Dickeya and Pectobacterium species, cause economically important diseases on vegetables, horticultural and other crops worldwide, resulting in high economic losses. Dickeya oryzae is the causal agent of bacterial heart rot, fruit collapse of pineapple, and bacterial foot rot of rice, and significantly reduces crop production. This study used the Multi-Locus Sequence Typing (MLST) method to define the phylogenetic relationships among the strains isolated from different sources, including kale, pineapples, corn, taro, and irrigation water. The housekeeping genes, dnaA, gapA, gyrB, atpD, and purA were selected after in silico screening for more SNPs and resolution capacity. A total of 119 strains of Dickeya and Pectobacterium species were evaluted. The phylogenetic and ClonalFrame analyses of the concatenated genes (total length ~4,625 bp) demonstrated that the pineapple strains from Hawaii were grouped with D. oryzea. The strains found associated with kale and taro presented close relationships with the D. zeae strains. ClonalFrame outcomes indicated 1.4- and 1.6-times higher recombination impact than point mutation (r/m 1. 364473 and 1. 576925) for D. zeae and D. oryzae data sets, respectively, and both species evolved from the same ancestor. Additionally, 24 selected strains of Dickeya and Pectobacterium species were compared to determine the main virulence factors, carbon utilization, and the ability to cause disease to different hosts. Tested Dickeya and Pectobacterium strains exhibited higher activities of cell wall degrading enzymes and motility at 28 °C. The kale strain (PL47) exhibited a distinct pattern of carbon utilization among Dickeya species. The Hawaiian taro strain showed the highest host specificity with taro from where it was originally isolated. We aimed to understand the genomic constituents of diverse D. zeae strains through comparative genomic analyses. We used Pacific Biosciences SMRT sequencing to sequence two high-quality complete genomes of novel strains of D. zeae: PL65 (size—4.74997 MB; depth—701x; GC—53.3%) and A5410 (size—4.7792 MB; depth—558x; GC—53.6%) isolated from economically important Hawaiian crops, taro, and pineapple, respectively. The genomic analyses indicated truncated type III and IV secretion systems in the taro strain and showed high heterogeneity in the type VI secretion system. Our study highlights genetic constituents of pathogenicity determinants and genomic heterogeneity that will help understand the virulence mechanisms and aggressiveness of this plant pathogen. We recently isolated the soft rot bacteria associated with kale and post-harvest taro, and both are economically important crops for Hawaii and the United States. The isolated strain (PL47) from kale showing soft rot symptom was identified as Dickeya zeae, and three strains (PL64T, PL63, and PL48) were identified as Pectobacterium, but the species designation of these strains were unclear. The pectinolytic bacterial strain PL65T isolated from an infected taro corm was identified as Dickeya zeae, but the species designation of this strain was unclear. The pectinolytic bacterial strains PL65T, isolated from an infected taro corm, PL64T, PL63, and PL48, isolated from infected kale, were subjected to polyphasic analysis to determine genomic phenotypic characteristics. Two representative strains, PL64T and PL65T were used for high-quality complete genome studies. The whole genome was sequenced using the Oxford Nanopore MinION and the Illumina Next Genome Sequencing (NGS) system and Pacific Biosciences SMRT. Multi-locus sequence analyses (MLSA) revealed strains PL64T and PL65T were in a novel clade separated from the other Dickeya zeae and Pectobacteium brasiliense strains, respectively. Phylogenetic analysis based on core gene sequences clearly showed two potentially new species within the Pectorobactericeae family. The in-silico DNA– DNA hybridization value of strain PL65T with other type strains of Dickeya species was <68%. Average nucleotide identity (ANI) analysis revealed that PL65T was at the margin of the species delineation cut-off values with a 96% ANI value. in-silico DNA–DNA hybridization value of strain PL64T with other type strains of Pectobacterium species was <60.2%. Average nucleotide identity (ANI) analysis revealed that PL64T was at the margin of the species delineation cut-off values with a ~94% ANI value. Based on the results of polyphasic approaches, including genome-to-genome comparisons presented in this report, we propose the establishment of two new species, Pectobacterium hawaiiense sp. nov. with PL64T as the type strain and Dickeya colocasia sp. nov. with PL65T as the type strain. We aimed to develop a rapid, field-deployable recombinase polymerase amplification (RPA) for specific and rapid detection of Dickeya spp. using lateral flow strips. A unique genomic region (mglA/mglC genes) conserved among Dickeya spp. was used to design highly specific, robust primers and probes for an RPA assay. Assay specificity was validated with 34 strains from Dickeya spp. and 24 strains from other genera and species; no false positives or negatives were detected. An RPA assay targeting the internal transcribed spacer region of the host genome was included to enhance the reliability and accuracy of the Dickeya assay. The detection limit of 1 fg was determined by both sensitivity and spiked sensitivity assays; no inhibitory effects were observed. The developed RPA assay is rapid, highly accurate, sensitive, and fully field deployable. It has numerous applications in routine diagnostics, surveillance, biosecurity, and disease management.
Characterization Of Mite-associated Viruses In Plant Pathosystems Of Hawaii
(University of Hawaii at Manoa, 2021) Olmedo Velarde, Alejandro; Melzer, Michael J.; Tropical Plant Pathology
Although mites' ability to vector plant viruses was demonstrated long ago, there remains a significant knowledge gap for these viruses and their interactions with their plant host and mite vector compared to other arthropod-vectored plant viruses. Most mite-transmitted plant viruses are vectored by mites that belong to the superfamily Eriophyoidea and the genus Brevipalpus (Acari: Tenuipalpidae). Viruses transmitted by eriophyid mites belong to at least seven genera, but Emaravirus members (family Fimoviridae) are considered emergent plant viruses of agricultural importance. The viruses transmitted by brevipalpus mites, also known as Brevipalpus-transmitted viruses (BTVs), belong to three genera classified within two families, Dichorhavirus (family Rhabdoviridae) and Cilevirus and Higrevirus (family Kitaviridae). The goal of this study is to lessen the knowledge gap for this group of plant viruses by further characterizing the genomics and biology of new and existing mite-associated viruses. In this work, we identify passionfruit (Passiflora edulis) as a new host for the BTV citrus leprosis virus C2 (genus Cilevirus). Also, after more than 60 years of absence, we report citrus leprosis in the US and determined it is caused by an orchid strain of the BTV orchid fleck virus (genus Dichorhavirus). This discovery has led to an eradication effort for this federally-actionable pest. A new BTV, named hibiscus yellow blotch virus (HYBV), was characterized, and provisionally placed within the genus Cilevirus. HYBV represents an evolutionary lineage between cileviruses and higrevirus and was detected in Brevipalpus yothersi, representing this new virus's potential vector. New hibiscus and citrus isolates of hibiscus green spot virus 2 (HGSV-2; genus Higrevirus) were characterized and found to possess low genetic diversity. The foundation for a reverse genetics system for BTVs was established through the development of an infectious clone of HGSV-2 that was capable of establishing infection in several natural and experimental plant hosts. This represents the first infectious clone developed for a BTV and a critical tool for future research in this arena. Finally, the transmission of HGSV-2 was demonstrated using B. azores mites. This is the first report of B. azores, having the capability to transmit viruses, and confirms HGSV-2’s status as a BTV. The virome of populations of flat mites collected from several plant hosts from two Hawaiian Islands was found to be predominated by picornavirids. Kita and Kita-like viruses were also found and supported a possible evolutionary scenario in which kitavirids emerged from arthropod-specific viruses such as negeviruses. A partial sequence of a putative new cile-like virus was found in tenuipalpid mites collected from pineapple. This virome study warrants further examination of flat mite populations from different hosts and geographic origins to surveil for and understand the diversity of BTV and BTV-like agents. Finally, ti ringspot-associated virus (TiRSaV) was characterized and found to contain at least five genomic segments, which could be detected in symptomatic ti plants (Cordyline fruticosa) only. TiRSaV was also detected in two putatively novel species of eriophyid mites recovered from symptomatic plants.
Microbial Associations in the Coffee Berry Borer, Hypothenemus hampei (Ferrari) (Coleoptera: Curculionidae: Scolytinae)
(University of Hawaii at Manoa, 2021) Aoki, Sayaka; Airf, Dr. Mohammad; Tropical Plant Pathology
Coffee (Coffea arabica and C. canephora; Rubiaceae) is one of the most important agricultural commodities in tropical and subtropical regions in the world, generating industries that surpass an estimated US$170 billion annually. The coffee berry borer (CBB), Hypothenemus hampei, is the most devastating insect pest of coffee worldwide. The insect was first reported in 1901, and it has invaded most coffee-producing countries, causing severe economic losses surpassing more than US$500 million annually. The coffee berry borer was first reported on Kona, island of Hawaii in 2010, and by 2020, it had been reported on Oahu, Maui, Kauai, and Lanai, causing significant economic loss in the coffee industry in Hawaii. Determining how the coffee berry borer became a coffee pest has been an area of interest due to the fact that it is the only insect to consume the coffee seeds inside the berry. Survival on the seeds therefore implies a mechanism to degrade caffeine. Insects have evolved a diversity of strategies to overcome challenges imposed by plants. One of the strategies to mitigate these challenges is to establish mutualistic associations with symbiotic microorganisms that could enable insects to thrive and reproduce within the unfavorable environments. These microbial symbionts associated with insects play pivotal roles in host survival, reproduction, host metabolism, and affect hosts’ biology and phenotypes via a multitude of functions, providing vital nutrients such as essential amino acids, nitrogen, vitamins, and sterols, breaking down cellulose and lignin materials that are hard to digest, influencing host plant usage, and mediating interactions with natural enemies. Recent studies have provided strong evidence of coffee berry borer-bacterial associations and the mechanisms, revealing the coffee berry borer has a range of associations with its bacterial symbionts for the survival and possibly affected its evolution and adaptations. Vega et al. (2002), revealed coffee berry borer associations with the maternally inherited bacterium Wolbachia from many countries. Ceja-Navarro et al. (2015) demonstrated that the coffee berry borer relies on caffeine-degrading bacterial symbionts in the alimentary canal in order to live in caffeine-rich conditions that are unfavorable for other insects. The caffeine-degrading microbiome reported by Ceja-Navarro et al. (2015) consisted of 13 bacterial species, and only one of these bacteria, i.e., Pseudomonas fulva, had the caffeine demethylase ndmA gene (methylxanthine N-demethylase A). When the insect was fed an artificial diet containing antibiotics, they lost the ability to degrade caffeine, and their reproductive fitness was negatively affected (Ceja-Navarro et al. (2015). Additional investigations on the role of bacteria on the fitness of the coffee berry borer are needed in order to better understand the biology of the insect. The objectives of this study are to: (1) Determine the identity of bacterial species associated with the coffee berry borer in commercial coffee farms and wild coffee in Hawaii; (2) Explore the transmission mode of caffeine degrading bacteria by visualizing the bacteria within the insect eggs by fluorescent in situ hybridization (FISH) targeting the caffeine demethylation gene (ndmA); (3) Isolate and identify bacterial species associated with the eggs and determine if they are capable of breaking down caffeine; and (4) Conduct whole genome sequencing to identify them into species-level, and understand genome biology of bacterial species isolated from CBB eggs.
Dissecting The Molecular Basis Of Basil-peronospora Belbahrii Interactions And Genetic Engineering For Disease Resistance
(University of Hawaii at Manoa, 2019) Navet, Natasha; Tian, Miaoying; Tropical Plant Pathology
Oomycetes form a distinct phylogenetic lineage of eukaryotic microorganisms capable of causing diseases in numerous plants and animals. Downy mildews are among the most devastating phytopathogenic oomycetes that form an obligate biotrophic relationship with their specific host. Peronospora belbahrii causes basil downy mildew (BDM), which is considered one of the most threatening diseases of cultivated basil globally. Sweet basil is the most popular herb used extensively in culinary, cosmetic, and therapeutic industry holding a high economic status. However, its production is in jeopardy due to BDM as disease resistance is lacking in sweet basil varieties and introgression of resistant genes through traditional breeding has faced several challenges. Limited fungicides have shown efficacy on BDM disease with a high risk of evolving fungicide resistant pathogen strains. The obligate biotrophic lifestyle of P. belbahrii and tetraploidy of sweet basil has hindered the study of basil-Peronospora belbahrii molecular-interactions due to lack of effective functional genomic tools. Using two Peronospora belbahrii genes with a putative role in pathogenesis and two sweet basil genes with potential functions in host resistance/susceptibility, studies in this dissertation established a set of functional analysis tools for basil and P. belbahrii studies. With these tools, the roles of the above genes in disease resistance/susceptibility were defined genetically, which offers initial insights into the molecular basis of basil- P. belbahrii interactions. In addition, multiple lines of downy mildew resistant sweet basil plants were generated with potential use in commercial production. Chapter 2 describes the functional characterization of a sweet basil L-type lectin gene, Oblectin 1. Plant lectins are carbohydrate-binding proteins, many of which have been shown to play essential roles in plant immunity. Oblectin 1 expression was found highly induced in a resistant cultivar during infection by P. belbahrii, but not detected in a susceptible one, suggesting its role in resistance to BDM. Transgenic plants ectopically expressing Oblectin 1 in a susceptible cultivar Genoveser were successfully generated through Agrobacterium-mediated transformation with high transformation efficiency. Homozygous transgenic lines conferred partial resistance to P. belbahrii. Through this objective, we developed a highly efficient basil transformation system and genetically defined the role of Oblectin 1 in resistance against P. belbahrii. Chapter 3 demonstrates the applicability of using host-induced gene silencing (HIGS) approach to decipher the role of two candidate pathogenicity-related genes, PbEC1 (Peronospora belbahrii Effector Candidate 1) and PbORCER1 (Peronospora belbahrii Oomycete RxLR-Containing Endoplasmic reticulum Resident 1). HIGS relies on an RNAi-based mechanism by which in planta generated small-interfering RNAs corresponding to a pathogen gene are transmitted to the pathogen during infection via cross-kingdom movement to silence the targeted gene. The candidate genes selected to test the efficacy of HIGS in basil were on the basis of effector features and sequence uniqueness. Transgenic basil plants, expressing inverted repeats of either PbEC1 or PbORCER1 partial coding-sequence flanking a pyruvate orthophosphate dikinase (PDK) intron, were generated. Upon infection, silencing of PbORCER1 was observed in all tested homozygous lines expressing RNAi constructs of PbORCER1, however, silencing of PbEC1 transcripts on homozygous transgenic plants expressing the PbEC1 RNAi construct was not consistently observed likely because its expression was highly induced during infection, which masked the detection of silencing. The pathogen growth on homozygous transgenic plants expressing either RNAi construct was markedly reduced. The results suggest that HIGS is operational during sweet basil-P. belbahrii interactions, and therefore offers a promising tool for functional genomics studies of P. belbahrii and generation of basil downy mildew disease resistance. Chapter 4 illustrates the establishment of an efficient Agrobacterium-delivered CRISPR/Cas9 system for targeted mutagenesis of a basil candidate susceptibility gene, ObDMR1. Two CRISPR/Cas9 constructs were developed, targeting different sites of the ObDMR1 gene. 92% of the transgenic plants displayed successful mutagenesis of ObDMR1 at one target site. ObDMR1 editing in sweet basil was shown to be predominantly heterozygous, but a complete knockout of all alleles was successfully achieved in one line in the first generation of transgenic plants. Transgene-free homozygous plants were obtained in the second generation. Homozygous plants having 1-bp frameshift mutations displayed dwarfism at the early seedling stage but later showed normal growth and development resembling wild type. In addition, knockout of ObDMR1 compromised basil downy mildew susceptibility. Collectively, our data in this chapter demonstrates the success of using CRISPR/Cas9-mediated gene editing to generate complete gene knockout basil mutants, the ease in obtaining transgene-free mutant plants in the successive generation, the development of basil downy mildew resistant plants, and the role of ObDMR1 in basil downy mildew disease development. These results contribute to the acceleration of basil gene functional analysis, molecular breeding, and development of resistant varieties for commercial use.
Management of Postharvest Decay of Tomato Fruit with Inorganic Salts and Natural Products
(University of Hawaii at Manoa, 2018-05) Ahmed, Firas A.; Tropical Plant Pathology
Gray mold caused by Botrytis cinerea and soft rot caused by Pectobacterium carotovorum are two of the most devastating pre- and postharvest diseases of tomato worldwide. A survey of fresh-market tomato fruit was conducted to determine the fungal and bacterial pathogens most commonly associated with postharvest disease on Oahu, Hawaii. The survey confirmed that gray mold and soft rot were the most common postharvest diseases of tomato in Hawaii. Pathogenicity tests revealed that 33 of 99 fungal isolates and 10 of 17 bacterial isolates were pathogenic on tomato varieties known as common market, cherry, and grape tomato. Based on fruit assays one fungal isolate, Botrytis cinerea (B03) and one bacterial strain, Pectobacterium carotovorum (BA17), were selected because both produced consistent symptoms and were highly virulent. As consumer-acceptable options for postharvest disease management are inadequate for gray mold and soft rot, experiments were designed to evaluate possible control measures for these two diseases. Effects of eleven natural products on spore germination and mycelial growth of Botrytis cinerea (B03) were evaluated using multi-well microplates and inhibitory assays, respectively. Capsicum chinense cv. Datil, C.annuum cv. Carnival, and an Agrichem Proprietary Formulation (APF) completely inhibited fungal germination at all evaluation times. Treatments with 40% plant extracts increased the generation of intercellular reactive oxygen species and the plasma membranes of fungal conidia were damaged. Fungal spores exposed to 40% plant extracts of C. chinense or C.annuum or 1 ml/L APF showed distinct signs of deterioration, deformation and condensed cytoplasm as observed using transmission electron microscopy. Capsicum chinense and C.annuum applications at 60% completely inhibited lesion development on tomato fruit following immersion for 10 min. An inorganic salt, potassium tetraborate tetrahydrate (B4K2O7.4H2O) (PTB), was evaluated for effects on the growth of P. carotovorum using strain BA17. Complete inhibition of bacterial colony development was achieved by treatment with PTB at 100 mM both at pH 9.2 and after adjustment to pH 7.0. Bactericidal activity was quantified and validated by counting fluorescently-labeled live and dead bacterial cells using flow cytometry, and reconfirmed using qPCR with high-affinity photoreactive DNA binding dye propidium monoazide (PMA). The results of flow cytometry, qPCR, and culturing confirmed that bacterial cells were killed following exposure to PTB at 100 mM. Bacterial cell membranes were damaged following a 5-min treatment and extrusion of cytoplasmic material from bacterial cells was observed using electronic transmission microscopy. Soft rot incidence on inoculated tomato fruit was reduced by dipping infected fruit in PTB at 100 mM for 5 min and no lesions developed following a 10-min treatment. PTB and APF were assessed as a preharvest spray in the greenhouse for reducing gray mold of tomato fruit caused Botrytis cinerea. PTB and APF were applied at three ripening stages: turning, light red, and pink. Both compounds reduced the disease severity of gray mold in greenhouse tomatoes when applied at the turning stage when fruit were stored either at 4 or 25˚C. In this study, recombinase polymerase amplification (RPA) was used for specific and rapid detection of Pectobacterium using lateral flow strips. The assay also included tissue from host plants (tomato and potato) as an internal control to enhance the reliability and accuracy of the assay. The assay readily distinguished Pectobacterium from other bacterial genera and proved to be an efficient assay for identification of the target pathogen in plant tissues. In summary, APF and PTB show promise for reducing gray mold as preharvest applications in the greenhouse tomato. In contrast, PTB is an effective alternative to other bactericides and antibiotics for controlling soft rot disease of tomato as a postharvest practical application.
Screening genes encoding transcription factors associated with pathogenesis in alternaria brassicicola
(University of Hawaii at Manoa, 2013-05) Srivastava, Akhil
The necrotrophic fungus Alternaria brassicicola causes black spot disease of brassicaceous plants, including green cabbage (Brassica oleracea) and the oil-producing B. napus. Pathogenesis is a multistep process that includes germination, penetration and colonization of host tissues, and survival structure formation. The de novo synthesis of various catabolic enzymes and secondary metabolites needed during each step in pathogenesis are under the regulation of transcription factors. Zinc finger DNA-binding domains containing transcription factor forms the largest family of transcription factors in eukaryotes. Based on the zinc binding motifs transcription factors are of three classes: Cys2-His2, Cys2-Cys2, and the fungal specific, binuclear Zn(II)2Cys6His6. In the present study we made knock out mutants of all 184 C2H2 Zinc finger transcription factor encoding genes by targeted gene disruption and 35 Zn(II)2Cys6His6 motif containing transcription factors through targeted gene deletion/replacement in Alternaria brassicicola. Our bioassays on detached leaves of green cabbage have identified twelve genes associated with early and late stages of plant infection. Among these twelve genes, two genes are pathogenicity factors as their mutants were nonpathogenic, while one showed increase in pathogenicity by almost 100%. Remaining nine genes were strong virulence factors whose mutants showed 50-90% reduction in disease symptoms compared to the wild type. We also discovered a unique gene whose mutants showed complete loss of conidia yet no effect on pathogenesis. We further report a gene encoding transcription factor associated with detoxification of phytoalexins, which is important for early plant colonization. All the discovered pathogenesis associated genes were novel pathogenicity or virulence factors and only one gene (PacC) was previously identified as a pathogenicity factor in other fungal species. The molecular mechanisms of pathogenesis and their regulation in necrotrophic fungi are in an early stage of research. This study sets the platform for discovery of downstream genes associated with pathogenesis and the characterization of their functions. Currently available data from this study indicate the importance of transcription factors as regulators of pathogenesis and as future targets for disease management.
Study of koa wilt disease: characterization of Acacia koa and Fusarium oxysporum
(University of Hawaii at Manoa, 2011-12) Shiraishi, Ayami
Koa (Acacia koa) is a highly valuable tree species in Hawaii. Over the past few decades however, the species has suffered from a severe wilt and dieback disease. The goal of this study is to manage the disease and improve koa plantation health. Fungal species were isolated from koa trees showing typical dieback symptoms, and species of these isolates were identified. Fusarium pseudocircinatum was recovered from the samples and its virulence to koa was confirmed with Koch's postulates. This is the first report of this fungal species in Hawaii. The population structure of F. pseudocircinatum and F. oxysporum collected from the samples were analyzed with pathogenicity tests, vegetative compatibility group (VCG) tests, and Amplified Fragment Length Polymorphism (AFLP) analyses. Forty-six isolates including F. oxysporum and F. pseudocircinatum were grouped into 16 VCGs. Among the highly virulent isolates, 86% belonged to the single VCG group, and these isolated clustered together in AFLP analyses. VCG and AFLP could be used for detection and identification of F. oxysporum strains in soils, and therefore would help koa growers to establish new koa plantations. VCG2 of F. oxysporum is a significant biological entity for which the name F. oxysporum f. sp. acaciae is proposed to reflect its virulence on koa. Pathogenicity tests conducted on 18 koa families revealed genetic variations in wilt tolerance among koa families. Mortality rates of the 18 families inoculated with a highly virulent F. oxysporum f. sp. acaciae isolate ranged from 0 % to 100 %. However, a strong interaction, P < 0.0001, was observed when host-pathogen interaction among five koa families and ten F. oxysporum f. sp. acaciae isolates were tested. This result indicates that each koa family and F. oxysporum isolate behave differently depending on their host family/pathogen combination. When koa mortality data from the field trials and pathogenicity tests were compared, no direct correlation was observed (correlation coefficient =-0.013). Understanding the host-pathogen relationship between koa and F. oxysporum and trends in koa mortality in the field trials will ultimately lead to the establishment of more effective disease screening protocols for koa silviculture in Hawaii.
Ecologically-based nematode management: exploiting nematode survival strategies for developing novel cover cropping and soil solarization practices
(University of Hawaii at Manoa, 2011-08) Marahatta, Sharadchandra Parasar
Improving the use of cover crops, such as Crotalaria juncea and Tagates patula, is needed for management of plant-parasitic and free-living nematodes. These cover crops might improve soil health conditions through a strip-till cover cropping practice. The integration of cover cropping of C. juncea and soil solarization could further improve ecological-based nematode management more than C. juncea alone. Additional improvement could be achieved if T. patula or C. juncea are found to suppress Meloidogyne incognita or Rotylenchulus reniformis more efficiently when these nematodes are in a vulnerable state. Strip-till cover cropping (STCC) of C. juncea suppressed M. incognita, but T. patula only suppressed M. incognita when planted immediately following after a susceptible host. In addition, STCC of C. juncea also enhanced population densities of bacterivorous and fungivorous nematodes, whereas STCC of T. patula enhanced these free-living nematodes inconsistently. Integration of C. juncea cover cropping and soil solarization did not reduce R. reniformis opulation densities, but reduced weed coverage as compared to C. juncea alone. The benefits of integrating C. juncea to soil solarization were only observed where C. juncea biomass was high (3.6 Mt/ha). Leachate of T. patula did not suppress egg hatch and J2 activity of M. incognita. However, planting of T. patula in a greenhouse pot experiment suppressed M. incognita J2 activity if the soil was conditioned with irrigation or cucumber leachate but not with dry treatment. On the other hand, planting of C. juncea and cowpea (Vigna unguiculata) reduced the number of R. reniformis in anhydrobiotic state. Amending soil with C. juncea suppressed R. reniformis more efficiently if fewer anhydrobiotic R. reniformis were present. In conclusion, C. juncea outperformed T. patula in a STCC system, whereas integration of C. juncea with soil solarization did not improve the benefit of a cover crop, C. juncea. Based on the greenhouse studies, the nematode suppressive effect of T. patula could be improved by planting T. patula immediately after a susceptible host. Benefits of C. juncea amendment were more obvious if used immediately after nematode susceptible (poor or good) hosts.
Developing a qPCR-based molecular technique for nematode community analysis
(University of Hawaii at Manoa, 2012-05) Wang, I-Chin
Nematodes are good indicators for soil health. However performing nematode community analysis is laborious and technically challenging. This research seeks to develop a qPCR-based molecular tool for nematode community analysis. qPCR detecting 18S rDNA offered one approach to identify and quantify free-living nematodes. Owing to too many unpredictable nematode genera across soil ecosystems, one strategy is to develop universal qPCR markers selective for key nematode guilds (Ba1, Ba2, F2, P4, Om4, Om5, and P5) that are most critical for nematode faunal analysis. Universal qPCR primers were successfully being identified for all of these guilds except for Ba1. Two primers were needed for F2; Om4, Om5, and P5 cannot be differentiated and were thus being combined as Om4/ Om5/ P5 by one primer. These primers were then verified by BLAST and then run through artificial nematode mixture sample composed with known nematode guilds. The results confirmed the validity of these universal primers. The next logical step was to run these qPCR to nematodes collected from four natural ecosystems: forest, organic, pineapple field, and beach sites. Visual nematode identification on these four systems was being conducted to compare results. Two qPCR standard curves (plasmid DNA and genomic DNA) were used to obtain nematode abundance of the four ecosystems. Since both DNA standard curves did not estimate nematode abundance comparable to the visual count, ranking of nematode community indices of the four ecosystems were compared between molecular and the visual methods. While the ranking calculated by the plasmid DNA standard curve of qPCR assay were not consistent with most of the nematode community indices calculated by visual method, 4 out of 8 nematode indices estimated by the gDNA standard curve were relatively consistent. This research provided universal nematode guild qPCR primer sets and initial protocol of qPCR-based molecular tool for soil nematode community analysis. Further research need to be conducted on better estimation of nematode abundance, richness and diversity. More universal primers selective for Ba1, Ba3, F3, P3, also Om4, Om5, and P5 individual primers are needed.
Characterization and management of different Fusarium species associated with orchids cultivated in Hawaiʻi
(University of Hawaii at Manoa, 2014-05) Srivastava, Shikha
Commercially grown orchids are among the most valuable ornamental crops produced in the United States. Hawaii is one of the three largest orchids-growing states in the nation, producing the greatest variety of tropical species and unique intergeneric hybrids. A decrease in orchid production has been observed in Hawaii in association with spot and blights of shoots and flowers, as well as root rot that result in rapid death of juveniles and slow decline of mature plants. Such decline is now commonly observed in nearly all nurseries across the Oahu and the Island of Hawaii. Fungal pathogens are speculated to be one of the important sources of diseases in orchids in Hawaii. In the present study 16 fungal genera were isolated from 60 plant samples collected from Oahu and the Island of Hawaii. Fusarium was the most prevalent genus, with 78% of the isolates and frequently associated with several orchid cultivars, including over 9 genera and hybrids. Eleven Fusarium species, namely F. proliferatum, F. oxysporum, F. solani, F. subglutinans, F. poae, F. begoniae, F. bulbicola, F. anthophilum, F. denticulatum, F. circinatum and F. semitectum were found. Fusarium oxysporum was identified and was the most commonly isolated species from all the samples followed by F. proliferatum and F. solani. All Fusarium species were tested for pathogenicity on four orchid genera. Among the 11 Fusarium species, 6 were found pathogenic on 4 orchid genera. Three species, F. proliferatum, F. oxysporum and F. solani, showed moderate to high virulence on Dendrobium, Cymbidium and Miltonia. Fusarium circinatum and F. poae were low to moderate virulent on Dendrobium and Cymbidium whereas F. begoniae caused very low virulence on Dendrobium and Miltonia. Cattleya was susceptible to F. oxysporum only. Pathogenicity assays of Fusarium isolates on different cultivars of orchids resulted in identification of pathogenic Fusarium species was followed by testing efficacy of three fungicides, Pyraclostrobin, a.i. at 25%, Azoxystrobin a.i. at 50% and Triticonazole, a.i. at 20% on mycelial inhibition on four most prevalent Fusarium species namely-F. proliferatum, F. oxysporum, F. solani and F. subglutinans. Triticonazole was the most effective fungicide in the in vitro tests and was further evaluated for its efficacy in on Dendrobium plants in vivo against the most aggressive species of Fusarium, F. proliferatum under greenhouse conditions. This dissertation provides a thorough study to identify pathogenic Fusarium species on orchids and provides information regarding pathogenicity on orchid genera other than Dendrobium, and viable chemical methods to manage Fusarium species on orchids in Hawaii.
Further characterization and detection of pineapple mealybug wilt associated viruses (PMWAVs)
(University of Hawaii at Manoa, 2014-12) Dey, Kishore Kumar
Mealybug wilt of pineapple (MWP) is one of the most destructive diseases of pineapple worldwide. A complex of pineapple mealybug wilt associated viruses (PMWaVs), all of which belong to the genus Ampelovirus (Family: Closteroviridae) have been identified in pineapple growing regions throughout the world. Three of these viruses, PMWaV-1, PMWaV-2, PMWaV-3 have been characterized. The disease etiology of MWP is still not completely resolved. To better understand the complex etiology of mealybug wilt of pineapple (MWP), it is essential that the genomes of PMWaVs be functionally characterized. In Hawaii, PMWaV-2 has an important role in the etiology of MWP, causing severe wilt symptoms and yield reductions in the presence of mealybug feeding, whereas infection by PMWaV-1 alone, with or without mealybug feeding, may cause some yield reduction but does not produce wilt symptoms. Higher plants use RNA silencing to defend against viral infections. As a counter defense, plant viruses have evolved proteins that suppress RNA silencing. In this study selected open reading frames (ORFs) of PMWaV-1 and PMWaV-2 were screened for their local and systemic suppressor activities in Agrobacterium-mediated transient assays using green fluorescent protein (GFP) in Nicotiana benthamiana. Results indicate that PMWaV-2 utilizes a multiplecomponent RNA silencing suppression mechanism. Two of the encoded proteins analyzed, p20 and CP, target both local and systemic silencing in N. benthamiana, whereas p22 and CPd targets only systemic silencing. In the related virus PMWaV-1, we found that only one of the encoded proteins analyzed, p61, had systemic suppressor activity. Of all the proteins tested from both viruses, only PMWaV-2 p20 suppressed local silencing induced by doublestranded (ds) RNA, but only when low levels of inducing dsRNA were used. None of the proteins analyzed showed an ability to interfere with the short distance systemic spread of silencing. We examined the mechanism of systemic suppression activity by investigating the effect of PMWaV-2-encoded p20 and CP proteins on secondary siRNAs. Our results suggest that PMWaV-2 p20 and CP (and probably other proteins) block the systemic silencing signal by repressing production of secondary siRNAs. We also demonstrate that PMWaV-2 p20 and p22 enhanced Potato virus X pathogenicity in N. benthamiana. The construction of a full-length and less than full length PMWaV-1 clone has been reported. The toxicity of certain region of the cloned PMWaV-1 genome in E. coli induced lethal mutations and re-arrangements, which might be the reason for its non-infectivity in N. benthamiana. A cell-free method, Circular polymerase extension cloning (CPEC) of creating infectious clone has been adapted successfully for the first time for a Potato virus X, plant virus. However technical refinement would require the making of an infectious clone of PMWaV-1. Considering the importance of PMWaV-2 in the disease etiology of MWP, a highly sensitive detection assay using a single closed tube nested PCR technique has been developed which can be used to detect very low PMWaV-2 titer in diseased plants and insects. The assay is made more flexible by inclusion of a Taqman® probe to make the assay quantitative.
Assessment of four soil nematode communities in Hawaii by different methods
(University of Hawaii at Manoa, 2008) Quintero, Tonia G.
Nematode communities are potentially excellent indicators of soil health. Assessing these nematodes communities using molecular and morphological techniques may seem straight forward. However, many challenges exist in implementing molecular techniques. First, molecular techniques employed to study nematode communities generally involve DNA extraction, PCR, cloning and molecular sequencing. Each of these steps can introduce bias into the analysis of a nematode community. Using morphology to identify and assess nematode soil health is cumbersome as well. Systematic knowledge of the nematode fauna is essential in order to assign nematodes to their appropriate classification. In new environments permanent slides and collaboration with laboratories that extensively work on nematode taxonomy for adequate identification of nematodes is essential. As a result, attempting to assess the health of soil should require multiple nematode faunal analyses over time which includes I) comparison of two methods, 2) Gap analyses and 3) extraction methods. It is believed that these experiments have provided pertinent information to help in the quest of measuring and monitoring soil health. However, there is not adequate information to confidently determine if the Manoa Falls Trail sites were "healthier" in comparison to the Whitmore site.

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