M.S. - Tropical Plant Pathology

Permanent URI for this collectionhttps://hdl.handle.net/10125/20044

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Exploring the potential of RNA interference technology for the control of phytophthora palmivora
(University of Hawai'i at Manoa, 2025) Topal, Okan; Sipes, Brent; Tropical Plant Pathology
Phytophthora palmivora is one of the most devastating plant pathogens of tropical crops, particularly on papaya and cacao in Hawaii, manifesting through symptoms such as fruit rot, stem rot and leaf damage. In papaya, traditional management methods involve the use of resistant cultivars, fungicides like phosphonates, metalaxyl, or copper hydroxide, and cultural practices. In addition to these conventional management techniques, double-stranded RNA (dsRNA) has emerged as a promising management approach, offering specificity in targeting mRNA sequences of the pathogenic genes of a pathogen while minimizing the environmental impact. Ppal15kDa, a protein secreted by P. palmivora, was previously shown to play an important role in the infection structure development and pathogenicity. In this thesis, the effect of Ppal15kDa dsRNA treatment on the pathogenicity of P. palmivora and development of infection structures was assessed using several assays. Co-incubation of fluorescein-labeled Ppal15kDa dsRNA and P. palmivora zoospores demonstrated the uptake of Ppal15kDa dsRNA by the zoospore cells. Experiments investigating the effect of Ppal15kDa dsRNA on germination tube length and appressorium formation showed that treatment with Ppal15kDa dsRNAs resulted in shorter germination tubes and reduced appressoria formation. Additionally, zoospores treated with Ppal15kDa dsRNA showed reduced lesion formation and smaller lesion diameters in papaya fruits and leaves. Lastly, Ppal15kDa dsRNA was shown to reduce the expression of Ppalk15kDa gene using reverse transcription quantitative PCR (RT-qPCR). Thus, this research demonstrated the promising effects of dsRNA treatment against P. palmivora on papaya.
INTERACTIONS BETWEEN THE FOODBORNE PATHOGEN (SALMONELLA ENTERICA SUBSP. ENTERICA SEROVAR ORANIENBURG AND ESCHERICHIA COLI O157:H7) AND SOFT-ROT PECTOBACTERIA (DICKEYA SPP.) ON ONIONS (ALLIUM CEPA): IMPLICATIONS FOR FOOD SAFETY
(University of Hawai'i at Manoa, 2024) Bhandari, Santosh; Dobhal, Shefali; Arif, Mohammad; Tropical Plant Pathology
Onions, one of the most consumed vegetables worldwide, is quite popular in United States, with an annual per capita consumption of 19.18 pounds and a total 2023 production of 51 million CWT (hundredweight). Despite their nutritional value and year-round availability, onions have been associated with multiple outbreaks of foodborne illnesses. The WHO estimates that contaminated food causes 600 million cases of foodborne diseases and 420,000 deaths globally each year. In the U.S., pathogens like Salmonella, Shiga toxin-producing Escherichia coli (STEC), and Listeria cause over 1.49 million illnesses annually, leading to thousands of hospitalizations and deaths. The irrigation water plays important role in causing foodborne outbreaks through the consumption of contaminated food including onions, where the foodborne pathogens get contaminated with the fresh produce when it comes in contact with irrigation water carrying foodborne pathogens such as Salmonella, E. coli.This research investigates the interplay between foodborne pathogens (FBP) such as Salmonella Oranienburg and E. coli O157:H7 and soft rot-causing pectinolytic bacteria (SRP), specifically Dickeya fangzhongdai and Dickeya dianthicola. Experiments were conducted to understand how SRP influences the survival, colonization, and population dynamics of these pathogens on onion slices (white, red, and yellow varieties) and onion plants.Key findings from the experiments indicate that SRP significantly enhances the proliferation of FBPs as significant difference was observed between the populations of S. Oranienburg in the presence and absence of SRP recovered from onion slices and white onion plants. For instance, at 7 dpi, the mean populations of S. Oranienburg were 4.24 ± 0.53 log10 CFU/g when inoculated alone, 6.70 ± 1.05 log10 CFU/g when co-inoculated with D. fangzhongdai, and 5.22 ± 0.50 log10 CFU/g when co-inoculated with D. dianthicola in white onion slices. Similarly, in yellow onion slices, the populations were 4.83 ± 0.78 log10 CFU/g, 7.39 ± 0.91 log10 CFU/g, and 5.96 ± 0.65 log10 CFU/g, respectively. In red onion slices, the population was significantly higher (5.98 ± 1.62 log10CFU/g) when co-inoculated with D. fangzhongdai at 7 dpi (p < 0.05) compared to inoculated alone (5.15 ± 0.40 log10CFU/g).In white onion plants, S. Oranienburg was not detected in most sections (roots, middle, crown, 50% plant height), except at the point of inoculation in a few plants. The mean population of S. Oranienburg was 8.52 ± 0.79 log10CFU/g and 9.40 ± 1.72 log10CFU/g in the middle section, and 8.52 ± 0.89 log10 CFU/g and 9.34 ± 1.49 log10 CFU/g in the crown region at 7 and 14 dpi, respectively. A significant difference was observed between the populations of S. Oranienburg in the presence and absence of SRP recovered from onion slices and white onion plants. At 7 dpi, the mean populations of S. Oranienburg were 4.24 ± 0.53 log10 CFU/g when inoculated alone, 6.70 ± 1.05 log10 CFU/g when co-inoculated with D. fangzhongdai, and 5.22 ± 0.50 log10 CFU/g when co-inoculated with D. dianthicola in white onion slices. Similarly, in yellow onion slices, the populations were 4.83 ± 0.78 log10 CFU/g, 7.39 ± 0.91 log10 CFU/g, and 5.96 ± 0.65 log10 CFU/g, respectively. In red onion slices, the population was significantly higher (5.98 ± 1.62 log10 CFU/g) when co-inoculated with D. fangzhongdai at 7 dpi (p < 0.05). In onion plants, S. Oranienburg was not detected in most sections (roots, middle, crown, 50% plant height), except at the point of inoculation in a few plants. The mean population of S. Oranienburg was 8.52 ± 0.79 log10 CFU/g and 9.40 ± 1.72 log10 CFU/g in the middle section, and 8.52 ± 0.89 log10 CFU/g and 9.34 ± 1.49 log10 CFU/g in the crown region at 7 and 14 dpi, respectively.Additionally, there was significant population of E. coli O157:H7 present in different sections (middle, crown and inoculation point) at 14 dpi in yellow (7.97±0.37 log10CFU/g, 8.12±0.57 log10CFU/g and 8.23±0.36 log10CFU/g), red (8.26 ± 0.29 log10CFU/g, 7.88 ± 0.85 log10CFU/g and 8.04 ± 0.68 log10CFU/g) and green onions (6.44 ± 0.45 log10CFU/g, 7.0 ± 0.36 log10CFU/g and 8.50 ± 0.18 log10CFU/g) when co-inoculated with D. fangzhongdai. Contrastingly, E. coli O157:H7 was not detected in any sections of yellow, red and green onion plants in absence of D. fangzhongdai from at 7 and 14 dpi. These findings highlight the role of SRP in facilitating the growth and internalization of foodborne pathogens in onions, raising concerns about their impact on food safety. Understanding these interactions is crucial for developing effective strategies to mitigate contamination risks and ensure public health. This study underscores the complex dynamics between plant and foodborne pathogens and their implications for agricultural practices and food safety.
Comparison of Entomopathogenic Nematode Efficacy via Infected Cadavers or Aqueous Applications for the Management of Cylas formicarius and Various Sweetpotato Insect Pests
(University of Hawai'i at Manoa, 2024) Silvester, Nicco Payne; Sipes, Brent; Tropical Plant Pathology
Entomopathogenic nematodes (EPNs) are deadly to certain insects and consequently have great potential as biological control agents. The potential of two local EPN isolates and their efficacy in the management of the sweet potato weevil, Cylas formicarius, was investigated. The development of a novel application method by releasing EPN-infected cadavers to improve the efficacy and ease of handling of EPN was investigated. Insect cadavers infected with EPN followed by slow desiccation of the cadavers to 85% relative humidity has been reported to protect the nematodes from ultraviolet radiation and desiccation, but the effects on the EPN are not known. Field experiments were established and aqueous applications of Heterorhabditis indica OM160 and Steinernema feltiae were made when the sweetpotato roots began to swell. Sweet potato weevil populations were low and little weevil damage occurred to the sweetpotato yield in the experiments, masking the efficacy of EPN treatments. In experiments comparing aqueous vs infected-cadaver applications of EPNs, sweetpotato yield was greater in plots treated with 2 cadavers of Galleria mellonella infected with H. indica OM160 compared to plots receiving 0, 3, or 6 cadavers (P<0.05). Desiccation of infected cadavers worked well with H. indica OM160 but S. feltiae MG14 migrated out of the drying cadavers. Heterorhabditis indica OM160 tolerated desiccation well with no effect on the nematode’s reproduction in the cadaver nor in the IJs’ ability to emerge and infect new insect larvae. IJs of H. indica OM160 in desiccated cadavers of G. mellonella were able to infect and kill 100%, 45% and 95% of Galleria mellonella, Oryctes rhinoceros and Tenebrio molitor larvae, respectively. Desiccated cadavers took on a “beef jerky” appearance and were not as fragile as non-desiccated cadavers. Desiccation of EPN-infected insect cadavers improves the ease of EPN handling for growers and decreases breakage of cadavers which can lower the efficacy of the EPNs.
Management of the sweet potato weevil (Cylas formicarius) using pheromone traps, entomopathogenic fungi, and entomopathogenic nematodes
(University of Hawai'i at Manoa, 2023) Wong, Landon Gee-Keong; Sipes, Brent S.; Tropical Plant Pathology
This research investigated the viability of three different entomopathogenic nematode (EPN) species from different genera, four strains of entomopathogenic fungi (EPF), and an Alpha Scents ® sweetpotato weevil trap for efficacy against the sweetpotato weevil (SPW). SPW mortality experiments using EPF and EPN were conducted in the laboratory. Heterorhabditis indica OM-160 had an adjusted mortality of 20-30%, Steinernema feltiae MG-14 had an adjusted mortality of 50-60%, while Oscheius tipulae OA-12 had an adjusted mortality at 30%. For EPF, Botanigard® (a.i. Beauveria bassiana GHA strain) caused a relative mortality above 30%, while mortality from Met Master® (a.i. Metarhizium anisopliae) was lower than the untreated control. Six field experiments were conducted to evaluate the efficacy of EPN and EPF. In five field experiments, application of commercial EPF products (Botanigard®, Mycotrol®, and Met Master® ) did not reduce SPW populations or damage. A Metarhizium isolated in Hawaii (KO002) substantially reduced damage and SPW population densities when inoculated into a compost and amended to soil at sweetpotato planting. S. feltiae MG-14 and O. tipulae OA-12 reduced SPW populations. EPN and commercial EPF effectiveness was negated when applied to the same plots. Pheromone traps were found to be effective at capturing males and monitoring population density of SPW in the field. Based on a field trial, SPW ppheromone traps did not interfere with biological control treatments. Therefore, for management of SPW, EPN and KO002 were effective against SPW. Commercial EPF products were ineffective and are not recommended. Pheromone traps were effective population monitors and are recommended for monitoring of sweetpotato weevil pest pressure.
Performance of conventional and biological fungicides for the management of coffee leaf rust and coffee anthracnose
(University of Hawai'i at Manoa, 2023) Buchholz, Elizabeth Rose; Cheng, Zhiqiang; Tropical Plant Pathology
Coffee Leaf Rust - Coffee leaf rust, Hemileia vastatrix., is a disease that has severely impacted the major coffee growing areas of the world. The presence of the uredinia reduces photosynthesis capabilities which will lead to premature leaf dropping and this defoliation can kill whole branches. If left untreated, yield can be reduced up to 50% in a growing season. The objective of this project is to collect efficacy and crop safety data to support selection and registration of fungicides that are safe on coffee and effective in the management of coffee leaf rust. In addition, there is an urgent need for systemic fungicides to provide sustained control of the disease throughout the year and to build a sound IPM program to manage fungicide resistance. Overall, Abound significantly reduced incidence of coffee leaf rust compared to the untreated control and Aproach® and Rally® provided good control compared to the untreated plots. Regalia® did not provide good control of coffee leaf rust compared to the control plots. Visual phytotoxicity ratings did not show any injury to coffee foliage, flowers, or berries. Anthracnose - Coffee is a high value commodity in Hawai‘i and diseases are affecting the overall health and productivity of the crop. Anthracnose, Colletotrichum spp., is a common yet easily overlooked problem facing the health of coffee fields across the islands. We sought to determine the efficacy of three fungicides on foliar and berry anthracnose infections. After an inoculation of the coffee trees with the naturally occurring Colletotrichum species, three fungicides were applied over the course of two months in efforts to control the disease. Data was collected in the following months to determine their effects on incidence and severity of disease on the foliage and berries of the coffee trees. Overall, Aproach® Prima significantly reduced incidence of anthracnose compared to the untreated control. Aprovia® Top and Cueva® did not provide good control compared to the untreated plots. Visual phytotoxicity ratings did not show any injury to coffee foliage or berries. More testing of these three products would be recommended to fully estimate their efficacy in controlling anthracnose on coffee.
Taxonomic and Functional Microbiome of Irrigation water and Ralstonia-Infected Ironwood (Casuarina equisetifolia) tree
(University of Hawaii at Manoa, 2022) Klair, Diksha; Arif, Mohammad; Tropical Plant Pathology
Microbiomics is an emerging field of research to identify the microbiota sharing the same niche, analyze the microbial genome, and unravel the interactions between the microbiome, host, and the environment, resulting in disease complexity. Also, the microbial community sharing the same niche maybe commensal, symbiotic, or pathogenic. With the advent of high-throughput DNA sequencing, independent of culture-based approaches allowed analysis of complex characteristics of the microbiome. This study focused on employing the next-generation sequencing platforms to investigate microbiomes of two ecosystems. The first ecosystem being the microbial structure and function of Hawaiian stream and spring water irrigation water systems, in addition to a technical comparison of employed technologies for microbial studies. The second ecosystem was to unravel the endophytic bacterial and fungal microbiota of ironwood trees, associated with Ralstonia infection, for deep insight into this complex and cryptic pathosystem. The need of studying the Hawaiian stream and spring water irrigation water systems is because of the water scarcity and a simultaneous need to increase food security. There has been a shift in agriculture irrigation systems from freshwater to alternative water sources such as reclaimed or recycled water. Irrigation water can be an important source of plant and food-borne pathogen contaminating plants and fresh produce, in addition to diverse unidentified bacterial consortia, agricultural waste, and antibiotic resistance genes (ARGs). The first objective of this study aimed to explore the bacterial microbiota from different irrigation water sources, and associated taro field water, from different geographical locations, in addition to investigating the function of the bacterial community discovered in the stream and spring water systems on Oahu, Hawaii. To analyze the bacterial community of 12 different irrigation water samples, high-quality DNA was isolated, sequenced for the V3-V4 region of 16S rRNA, full-length 16S rRNA, and shotgun metagenomic using Illumina iSeq100, Oxford Nanopore MinION, and Illumina NovaSeq100, respectively. Technical comparison of the short amplicon-based analysis provided a high resolution of taxonomic classification at the phylum level, whereas full 16S rRNA could validated the microbial classification of samples sequenced for short amplicon and shotgun metagenomic to the genus and species level. Functional comparison of stream and spring water revealed only 12% shared genes with a total of 95 ARGs with variable abundance. Therefore, this study provides a better understanding of the selection of appropriate sequencing platforms and pipelines to study irrigation water microbiome. Deciphering microbial communities and their functions will also be beneficial in the formulation of better disease management strategies. For the second ecosystem to be investigated, endophytic microbiota of the ironwood tree was the focus of the study. Ironwood is an important cultural and ecological component of the Island of Guam which has been subjected to different biotic (mainly Ralstonia infection) and abiotic stresses causing tree decline. However, little research is known about the complex Ironwood-Ralstonia pathosystem which could assist in managing the “disease complex”. The difficulty in studying this disease complex in the past was mainly the failure to isolate the causal agent, Ralstonia species, from symptomatic trees. Instead, other bacterial genera like Klebsiella and Pseudomonas were commonly isolated from infected trees. In addition, inconsistent wet wood symptoms, failure/inconsistent to prove Koch’s postulates, and incomplete knowledge about associated factors in disease development results in “disease complexity”. This study aimed to characterize the endophytic bacterial and fungal microbiota of 101 ironwood tree samples associated with Ralstonia infection and other factors, using amplicon-based approach targeting 16S rRNA and ITS gene region. Overall, Proteobacteria and Basidiomycota were the dominant bacterial and fungal phyla identified from the samples. Owing to the poorly annotated fungal database, a high number of reads remained unclassified for fungal classification. Whereas better resolution was obtained for bacterial genus classification, identifying Ralstonia in infected samples with higher accuracy. Samples infected with Ralstonia showed significantly lower bacterial richness and diversity, indicating ability of pathogens to impact endophytic microbial selection within the plant system. Furthermore, influence of Ralstonia was observed in affecting the fungal diversity and richness significantly in addition to Ganodermataceae infection and xylem conductivity. Based on this study, it is evident that deciphering the endophytic microbiota and knowing the role of associated biotic and abiotic factors can be advantageous to devise microbial bioformulations and develop efficient disease management strategies.
Genome Informed Comparative Characterization Of A Banana Fusarium Wilt Pathogen In Hawai’i And Screening Banana Germplasm For Disease Resistance
(University of Hawaii at Manoa, 2022) Shipman, Aaron Keith; Tian, Miaoying; Tropical Plant Pathology
Bananas are one of the most popular fruits, being crucial to global food and economic security. Fusarium wilt of banana, also known as Panama wilt, is a major threat to banana production worldwide. The disease is caused by several pathogens in distinct lineages of the Fusarium oxysporum species complex (FOSC). Fusarium oxysporum f. sp. cubense (FOC) refers to a polyphyletic group of FOSC pathogens that are specialized for attacking bananas, which all cause vascular wilt disease. Few lineages of FOC are well understood regarding evolutionary relatedness, virulence, and pathogenic host range on different types of bananas. A strain of the pathogen was recently isolated during a survey for Fusarium wilt of banana in the State of Hawaii. A comparative study was performed by whole genome sequencing and by evaluating the responses of a diverse panel of 26 banana species, subspecies, and domesticated varieties of banana. The Hawaii isolate was pathogenic to 18 of these accessions, of which 8 were highly susceptible to disease. ‘Gros Michel’, the banana variety differentiating race 1 pathogenicity was highly susceptible while, ‘Cavendish’, the race 4 differential was highly resistant. Very interestingly, ‘Bluggoe’ the variety differentiating race 2 pathogenicity was only moderately resistant. All the major banana cultivars grown in Hawaii were affected by this disease. Phylogenetic analysis of the TEF-1α (translation elongation factor), the RPB1 (largest subunit of DNA-dependent RNA polymerase II), and the RPB2 (second largest subunit of DNA-dependent RNA polymerase II) barcoding regions classified this Hawaii isolate to an understudied FOC lineage undergoing geographical range expansion and genetic diversification in Asia, which was proposed to be named Fusarium grosmichelii in 2019. More recent advances into the systematics of FOSC in 2020 suggested that all FOSC diversity should be classified into three phylogenetic species. Through further phylogenetic analysis based on seven genomic loci and the conserved coding sequences of whole genomes, multiple lineages of FOC were shown, including the Hawaii isolate, to be nestled within a larger species proposed as Fusarium oxysporum sensu stricto by the 2020 study, while other FOC lineages were classified into the two other species. This is the first study showing FOC is classified into the three species delimited within the Fusarium oxysporum species complex. Furthermore, whole genome phylogenies suggested that the lineage of Fusarium grosmichelii is more closely related to FOSC isolates pathogenic to pea and watermelon than to other lineages pathogenic to banana. Screening of a 99.7% complete de novo genome assembly as indicated by analysis with BUSCO software found a unique profile of secreted in xylem (SIX) genes, composed of two different copies of SIX1 and SIX9 each and a copy of SIX13, with SIX4 and SIX6 absent. This is the first report of this FOC lineage outside of Asia, and the first report of an isolate of this lineage lacking homologs of SIX4 and SIX6. This study also reports the first formal description of a pathogen found causing Fusarium wilt of banana in Hawaii, and to our knowledge, produced the first publicly available whole genome assembly representing this lineage of FOC. These findings are presented alongside the differential pathogenic host range and virulence of this Hawaii isolate on a large diverse set of banana germplasm, which the dual approach of de novo genome assembly and large disease assay is unavailable for most lineages. These findings serve to further basic knowledge on this lineage of FOC regarding pathogenicity and evolutionary systematics. The implications of this study could guide banana breeding and cultivar deployment in Hawaii and beyond.
Strategies to Enhance Efficacy of Entomopathogenic Nematodes against Diamondback Moth (Plutella xylostella) and Imported cabbageworm (Pieris rapae)
(University of Hawaii at Manoa, 2021) Budhathoki, Sabina; Wang, Koon-Hui; Tropical Plant Pathology
Diamondback moth (DBM), Plutella xylostella, and imported cabbage worm (ICW), Pieris rapae, are the most destructive insect pests of cruciferous crops worldwide. Whereas various control measures against ICW are viable, DBM management in Hawaii is challenged by the development of insecticide resistant populations. This thesis focuses on exploring IPM strategies to enhance the efficacy of entomopathogenic nematodes (EPN) against DBM and ICW. Three approaches compatible with organic farming were examined to enhance the efficacy of foliar application of EPNs by integrating EPN sprays with 1) trap cropping, 2) intermittent sprinkler irrigation, and 3) using adjuvants comply with organic certification. For the trap cropping approach, three 2 × 2 (trap crop × EPN) factorial field trials were conducted by using kai choi (Brassica juncea) as trap crop and Steinernema feltiae for EPN sprays supplemented with 1.6 ml/L (or 20 fl oz/acre) on head cabbage (Brassica oleraceae var capitata) and kale (Brassica oleraceae var acephala). On head cabbage, trap cropping by kai choi reduced the abundance of DBM by 46%, ICW by 73%, DBM damage by 45% and ICW damage by 33%, respectively. On the other hand, EPN reduced DBM number in trap crop plots and ICW in no trap crop plots only. Effects of trap cropping were less on kale compared to that observed on cabbage. In the first kale trial, trap crop suppressed 50% of DBM abundance and 19% of leaves with DBM damage, whereas in the second trial, trap cropping only reduced 13% of leaves with ICW damage. EPNs was not effective against number and damage of both ICW and DBM in Kale Trial I, but it suppressed DBM numbers by 100% in Trial II (P ≤ 0.05) soon after application. Overall, trap cropping did not improve the efficacy of EPN spray. EPN only suppressed DBM or ICW successfully when average pest pressure throughout a crop was below 0.5/plant. For the intermittent sprinkler irrigation approach, two 3 × 2 (sprinkler irrigation regime × EPN +/-) factorial-split plot cabbage field trials were conducted. The three main plots of intermittent sprinkler irrigation regimes were: 1) 5-min sprinkler irrigation twice at dusk (6:00 and 8:00 pm) (Sd), 2) 5-min sprinkler irrigation from 8:00 am to 4:00 pm at 2-hour intervals and twice at dusk (6:00 pm and 8:00 pm) (SDd), and 3) no sprinkler irrigation (NS). Each main plot was split into subplots of foliar EPN (1.25 million IJs/ha) or no EPN (EPN-) applications. In Trial I, SDd and Sd decreased DBM damage on head cabbage by 19% compared to the NS but they had no effect in Trial II. SDd also reduced ICW damage by 88% in Trial I and by 45% in Trial II. In terms of insect abundance, SDd reduced ICW numbers on head cabbage by 86% compared to the NS control in Trial I and by 58% in Trial II. However, SDd and Sd did not affect DBM numbers. Unfortunately, no interaction occurred between sprinkler irrigation and EPN application, indicating that intermittent sprinkler irrigation also did not improve the performance of EPN on these pests. To explore the third approach to improve EPN performance, two greenhouse experiments were conducted to test the efficacy of different adjuvants in enhancing the persistence of Steinernema feltiae MG-14 and its suppression against DBM. The adjuvant treatments tested were 3.9 ml/L (i.e. 50 fl oz/100 gal) of 1) Oroboost®, 2) Kinetic® and 3) Exit® compared to a 4) water control with EPN only where S. feltiae IJs were exposed to high heat in a greenhouse for 0, 30, 60, 120 and 180 min. Only Oroboost® extended the survival rates of S. feltiae for 2 hours. In fact, Kinetic® and Exit® showed a sign of toxicity against EPN. Oroboost® at 3.9 ml/L added to the EPN spray was more effective than Oroboost® at 1.6 ml/L (adjuvant rate used in the field trials as described above) in reducing the DBM population on cabbage plants 2 days after EPN application in a greenhouse. A laboratory and a greenhouse experiment were followed up to test dosages of S. feltiae at 0, 0.625, 1.25 and 2.5 IJ/cm2 mixed with Oroboost® at 3.9 ml/L against DBM larvae and pupae. Both experiments showed that 0.625 IJ/cm2 was as effective as the commercial (1.25 IJ/cm2 equivalent to 125 mil IJs/ha) and high (2.5 IJ/cm2 equivalent to 250 mil IJs/ha) dosages of S. feltiae in killing and infecting DBM. Interestingly, both larva and pupa of DBM were equally susceptible to S. feltiae infection. In summary, kai choi was more effective as a trap crop against DBM and ICW damage when intercropped with cabbage than with kale. Effect of kai choi as a trap crop in a kale cropping system was sporadic, mostly effective against DBM damage but not against ICW. The intermittent sprinkler irrigation (ISI) regimes tested in this thesis were only effective and rather significantly against ICW when ISI was in the daytime (> 80% in one trial and > 45% in another). ISI only reduced DBM damage by < 20% regardless of day or dusk ISI. More ISI regimes later at night need to be tested against DBM. The EPN efficacy tests conducted in the laboratory or greenhouse clearly showed that the high commercial EPN rate is not warranted but use of adjuvant is imperative. Future trap cropping field trials need to re-evaluate efficacy of S. feltiae with 62.5 mil IJs/ha supplemented with 3.9 ml Oroboost®/L. None-the-less, this project provided promising progress towards non-pesticide-based approaches against DBM and ICW that can help to mitigate insecticide resistance problem for organic farmers in Hawaii.
Viral Diseases Of Crotalaria In Hawaii
(University of Hawaii at Manoa, 2021) Kong, Alexandra; Melzer, Michael J.; Tropical Plant Pathology
Sunn hemp (Crotalaria juncea) is a leguminous cover crop valued for its ability torapidly accumulate biomass and fix nitrogen. In October 2016, farmers in Maui County noticed symptoms of leaf mottle, reduced seed pod numbers, and a reduction in seed yield of their sunn hemp crop. Although these symptoms are indicative of Sunn hemp mosaic virus (SHMV) infection, next generation sequencing of a dsRNA-based library revealed the presence of two viruses not yet reported in Hawaii. The first, Tobacco streak virus (TSV) is Ilarvirus previously reported in the continental United States, but not in Hawaii. The second virus is a novel viral species with notable homology to members of the genus Tobamovirus and most similar in sequence identity to SHMV. In 2018 sunn hemp samples from Poamoho with symptoms of leaf chlorosis were also sampled. Next generation sequencing of a dsRNA-based library revealed the presence of another virus not yet reported in Hawaii, the Fabavirus Broad bean wilt virus 2. A weedy sunn hemp relative, Crotalaria micans was also sampled from the Big Island and was found to be infected with the potyvirus Bean common mosaic virus. The presence of these new viruses could cause potential problems not only to the sunn hemp industry in Hawaii, but may potentially impact local agriculture. The purpose of this study was to identify the pathogen(s) responsible for the observed symptoms on C. juncea and other Crotalaria species as well as better characterize this new tobamovirus.
Evolutionary relationships and molecular diagnostics of Ralstonia solanacearum species complex associated with declining ironwood trees in Guam
(University of Hawaii at Manoa, 2020) Paudel , Sujan; Arif, Mohammad; Tropical Plant Pathology
Ironwood (Casuarina equisetifolia) is an important component of the island of Guam’s culture and ecology. Different biotic and abiotic factors have been associated with the declining ironwood trees on Guam. The bacterial wilt disease caused by Ralstonia solanacearum species complex (RSSC) was considered an associated factor after the positive test results of RSSC specific immunostrips with ooze from declining trees. The long-range spread, broad host range along with emerging new hosts of the bacterium necessitated the characterization of RSSC ironwood decline strains to understand the diversity, evolutionary relationships among the strains and predict the future movement pattern. In this study, we developed an efficient protocol for the isolation and characterization of RSSC strains from the declining trees. We also used the MLST approach to study the diversity, evolutionary relationships and predict the genealogies of the isolated strains. The presence of fast-growing saprophytes in the declining trees necessitated the development of field deployable and rapid Exo-RPA assay in our study. Furthermore, we also developed a five-plex multiplex PCR assay to detect and differentiate the genospecies of RSSC strains associated with ironwood and all other hosts including the Select Agent (SA) group. The isolation of RSSC from the declining ironwood tree was found to be highly effective with the use of modified SMSA media from the root slices. Both R. pseudosolanacearum (Rps) and R. solanacearum (Rs) were found to be associated with decline although the later species was found to be much lesser in number (3) compared to the former species (35). The phenotypic characterization assays showed the similar utilization pattern for the Rps strains and for the Rs strains. The ironwood Rps population in Guam was found to be highly clonal with least nucleotide diversity and contracting population structure. Our analysis showed North and Eastern Asia and Indonesia and Northern Australia region as the potential origin of ironwood Phylotype I strains whereas Central America and South-Eastern USA and Northern Latin America and Caribbean clade may be the potential origin of Phylotype II ironwood strains. The unique target genomic region for the molecular diagnostics of RSSC ironwood strains was determined using whole genome based comparative genomics approach and ORTHOMCL. The developed RPA assay was found to be highly specific, rapid, field- deployable, and efficient in detection of target directly from the infected plant material with least or no effect of plant inhibitors. When tested with a total of 110 RSSC strains from all the genospecies and SA group, the developed multiplex assay gave accurate results with no cross-amplification. The multiplex PCR assay also successfully differentiated the genospecies from the artificially inoculated plant DNA proving its high usefulness in culture characterization, routine diagnostics, and surveys. Microscopy, labelling studies and whole-genome based studies will provide better understanding about the role of secondary saprophytes in ironwood decline and unlock the questions about role of the bacterium as a primary or an opportunistic pathogen.
Foliar Nematode Control Using New Nematicide Formulations and Ornamental Plant Safety Associated with Several New Nematicides
(University of Hawaii at Manoa, 2019) Mitsuda, Kelsey; Cheng, Zhiqiang; Tropical Plant Pathology
Aphelenchoides fragariae is a species of foliar nematode that is an increasingly widespread pathogen of ornamental crops with a wide host range, attacking more than 250 plants species in 47 plant families. The most recognizable field symptom of foliar nematodes is the interveinal lesions on leaves. Previously, chemical treatments using active ingredients such as methyl bromide, oxamyl and parathion were effective against foliar nematodes. However, due to environmental concerns and their high toxicity, these chemicals are no longer available for foliar nematode control. The overall goal of this study is therefore to determine the effectiveness of several new, reduced-risk nematicides against foliar nematodes on certain popular ornamental plants in Hawaiʻi. Specific objectives are 1) the efficacy of several newly developed nematicides for managing foliar nematodes on the fern species Microlepia strigosa; and 2) if these newly developed nematicides have phytotoxicity effects on ornamental plants commonly used in Hawaiʻi's landscape industry; Microlepia strigosa, Frangipani, Raphiolepsis indica, Hibiscus, Phalaenopsis, and Anthurium adreanum. Foliar nematodes were extracted from infected fern tissues using the Baermann funnel technique. These nematodes were cultured in the lab using carrot discs and the cultures were refreshed every 5-7 weeks. New nematicides ESP 715 consisting of fluopyram as the active ingredient (a.i.) along with two other bionematicides, MBI 304 and Majestene, with a.i. of Chromobacterium spp. strain extract and Burkholderia spp. strain extract, respectively, were tested for potential control of Aphelenchoides spp. on Microlepia strigosa. Height, width and weight of fern were assessed weekly over 6 weeks after foliar nematode inoculation on the leaves. Foliar nematode damage was assessed at the end of the experiment. In addition, ESP 715, MBI 304 and Majestene were examined for phytotoxicity on M. strigosa, Frangipani, R. indica, Hibiscus, Phalaenopsis, and A. adreanum at various rates: Fluopyram at 0 ml/L, 0.33 ml/L, 0.66 ml/L and 1.34 ml/L. Except palapalai which was only tested with 2 rates of fluopyram: 0.66ml/L and 1.34 ml/L. Additionally MBI 304 and Majestene were examined for phytotoxiticy on M. strigosa plants: MBI 304 at 4,793 mg/L and MBI 205 at 20 ml/L. All plants treated with these nematicides received three applications at 14-day intervals. Untreated plants were included as the control. No visual foliar phytotoxicity symptoms were observed on all treatments throughout the 26-week evaluation period for Frangipani, R. indica, Hibiscus, Phalaenopsis, and A. adraeanum and the 14-week period for bionematicides on M. strigosa., except for fluopyram on M. strigosa. Fluopyram at both tested rates caused visual phytotoxicity effects. 0.66 ml/L of fluopyram caused severity ratings of 1.05 on the 0-5 scale. 1.34 ml/L of fluopyram caused severity ratings of 0.95. Severity ratings for both rates of fluopyram were significantly higher than the noninoculated control and significantly lower than the inoculated control. However, fluopyram did not suppress foliar nematodes. Burkholderia and Chromobacterium did not suppress the number of foliar nematodes significantly but reduced the numbers by 65.7% and 75.8%, respectively. Although various plant growth factors were stunted on hibiscus, orchid, anthurium, indian hawthorn and plumeria by fluopyram, it did not affect the marketability of the plants as no visual foliar phytotoxicity symptom was observed.
Survey For Ceratocystis Fimbriata On Syngonium Species, Host Range Test And Molecular Characterization Of The Isolates Collected
(University of Hawaii at Manoa, 2018-12) Dhakal, Upasana; Uchida, Janice; Tropical Plant Pathology
Rapid Ohia Death (ROD) caused by Ceratocystis lukuohia and C. huliohia is killing several thousand hectares of ohia forests in the Island of Hawaii. Ceratocystis lukuohia is closely related to C. fimbriata from Syngonium in Hawaii based on phylogenetic analysis. However, the distribution and diversity of the C. fimbriata from Syngonium is not known. Surveys were conducted on the Islands of Oahu, Hawaii, Maui, and Kauai to determine the distribution of C. fimbriata in Syngonium. Ceratocystis fimbriata was recovered from the samples collected from two commercial nurseries in Hilo, Hawaii. Ceratocystis fimbriata was not isolated from the samples collected from Oahu and no Syngonium plants having typical ¬¬¬symptoms of C. fimbriata were obtained from Maui and Kauai. Koch’s postulate was established on Syngonium with the C. fimbriata isolates collected from Syngonium. However, Ohia plants inoculated with C. fimbriata from Syngonium showed no symptoms after three months post inoculation even when the optimal conditions necessary for infections were provided. Ceratocystis fimbriata isolates 3401, 3459 and 3466 were not different to each other in terms of radial growth at 15°C, 20°C, 25°C and 30°C as revealed by analysis of variance at 0.05 level of significance but statistical analysis of the dimensions of perithecia, asexual and sexual spores at the same level of significance showed that C. fimbriata isolates 3401, 3421, 3459 and 3466 were significantly different . Fifteen C. fimbriata isolates collected from Hilo, Hawaii during the survey were clonal. They showed no sequence diversity for Internal Transcribed Spacer (ITS) rDNA region, Beta-tubulin 1(Bt1), Transcription elongation factor-1 alpha (tef1), Guanine Nucleotide binding protein subunit beta-like protein (ms204) and second largest subunits of RNA polymerase II (rpb2). Ceratocystis fimbriata collected from Syngonium in this study was identical for all five genes to C. fimbriata collected from Syngonium in the 1980s and these isolates grouped together in the phylogenetic tree with strong branch support.
Detection and Characterization of Plant Pathogenic Viruses in Hawaii
(University of Hawaii at Manoa, 2018-08) Green, James C.; Tropical Plant Pathology
Historically and currently virus diseases have caused significant loss to agricultural stakeholders in HI (HI) and worldwide. Pineapple was one of HI’s most important agricultural crops for many years though recently production has declined. Mealybug wilt of pineapple (MWP) is one of the most destructive diseases of pineapple worldwide and the causal agents are single-stranded RNA (ssRNA) viruses know as Pineapple mealybug wilt-associated viruses (PMWaVs) of the Ampelovirus genus of the Closteroviridae family. Though the disease etiology of MWP is not completely understood much of the genomes of the various PMWaVs have been characterized previously; this study furthers the characterization of several PMWaVs. To clarify the mysterious etiology of MWP it is important that the function of the genes of the various PMWaVs are well understood, underlying the importance of genome characterization. The genomes of PMWaV-1, PMWaV-2 and PMWaV-3 were first characterized in HI prior to this study, but only PMWaV-1 has been completely characterized. Two putative PMWaV member species: PMWaV-4 and PMWaV-5 had been reported from HI and Australia respectively. The putative PMWaV-4 was first reported in 2005. The PMWaV-4 heat shock protein 70 (HSP70) was the only gene reported. Prior to 2011, the criteria for species demarcation in the Ampelovirus genus was based off a 10% divergence of the amino acid (aa) sequence of the HSP70, RNA dependent RNA polymerase (RdRp) and coat protein (CP) genes; in 2011, the criteria was changed to a 25% divergence of the same three genes. In this study we characterize the remainder of the PMWaV-4 genome. Total RNA was used as the template for cDNA library construction and subjected to high-throughput sequencing (HTS), on an Illumina TruSeq 500 platform, multiple assembly pipelines were utilized for contig assembly and a 13,150 nucleotide (nt) scaffold was assembled that shares a high level of sequence homology with the 13,070 nt PMWaV-1 reference genome (AF414119). The aa similarity of the three species demarcation genes of PMWaV-4 compared to PMWaV-1 is 89, 87 and 85% for the RdRp, HSP70 and CP respectively. Based off the sequence similarity to PMWaV-1 we have determined PMWaV-4 to be a strain of PMWaV-1 rather than a distinct species. Additionally, HTS assemblies of PMWaV-1, PMWaV-2 and PMWaV -3 genomes shared a 99, 99 and 97%, respectively, nt similarity to their originally published genomes and supplemented the preexisting PMWaV-2 and PMWaV-3 sequences with 166 and 641 nt, respectively, in the 5’ termini of the viruses. The potyvirus Banana bract mosaic virus (BBrMV) and badnavirus Canna yellow mottle virus (CaYMV) were previously identified infecting Alpinia purpurata (flowering ginger) in HI. Recently, farmers growing flowering ginger have reported severe virus-like disease symptoms from multiple farms on Oahu, HI. Surveys were conducted in September 2016 and April 2018 molecular and serological techniques were used to assay symptomatic flowering ginger for the presence of the previously identified viruses. CaYMV was found to be widespread at all but one location however no BBrMV was detected in any samples from either survey. Characteristic symptoms of CaYMV and BBrMV were not observed, however streaking symptoms in CaYMV and BBrMV negative leaves, stems and bracts were observed. The lack of conclusive evidence linking CaYMV or BBrMV infection with the symptomatic flowering ginger assayed during this study may indicate the presence of another pathogen’s involvement. Further study is necessary to conclude the casual organism of the disease found on flowering ginger. Honolulu, HI is home to many community gardens which function to provide many urban dwellers the privilege of access to land for growing of edible and horticultural plants. Unfortunately, these areas are often ‘hotbeds’ of disease and pathogen dispersal. Recently, we identified several different plants infected with viruses from diverse crop and horticultural species. Potyvirus-specific ELISA detected potyviruses in symptomatic Passiflora spp. (passionfruit) and Phaseolus lunatus (lima bean). RT-PCR assay using degenerate potyvirus-specific primers then confirmed the ELISA results. Amplicons from the RT-PCR assay were cloned and sequenced using Sanger sequencing. Sequencing results revealed the potyviruses to be Watermelon mosaic virus (WMV) and Bean common mosaic virus (BCMV) infecting and passionfruit and lima bean respectively. WMV had previously been reported infecting passionfruit in HI. This was the first report of BCMV in HI and the sequence homology showed a high degree, 92% nt and 100% aa similarity to a BCMV isolate from China. A BCMV-specific ELISA was used to reconfirm the sequencing results. The unusually diverse amount of plants grown in a community garden as well as the high volume of individuals with access to the area provide the ideal circumstances for dissemination of plant pathogens.
Identification and Pathogenicity Test of Bacterial Symbionts from an Entomopathogenic Oscheius
(University of Hawaii at Manoa, 2018-08) Alhussaini, Abdulrahman A.; Tropical Plant Pathology
The main objectives of this thesis research were to examine Oscheius nematodes for its 1) associated bacteria (to be identified), 2) associated bacteria pathogenicity on insects, and 3) status as entomopathogenic nematodes. Nematodes are unsegmented pseudocoelomic roundworms that exist in all environments and occupy all trophic levels including of bacterial-feeders, predators, and parasites. Oscheius spp. are clearly bacterial-feeding nematodes but some isolates have behaviors with entomopathogenicity. A series of tests were conducted to isolate, identify, and determine insect toxicity of bacteria associated with several Hawaii isolates of Oscheius sp. Bacteria were isolated from Oscheius isolates BI 1a, BI 12a, OJ 4a, OJ 5b and compared with that of a well-recognized entomopathogenic nematode, Steinernema feltiae MG14. Eleven bacterial isolates were identified and isolated from four different Oscheius populations through DNA sequencing. Enterobacter genus was most commonly isolated followed by Pseudomonas and Enterococcus. Pathogenicity of Serratia sp., Enterococcus sp., and Pseudomonas sp. isolated were evaluated on mealworm (Tenebrio molitor) larvae using two assays: 1) feeding assay on a bacteria-inoculated diet, or 2) directly assay by injecting the bacteria into the hemolymph. All assays showed that these three bacteria isolates were pathogenic to the mealworms within 48 hour after the feeding or injection. Among the three bacteria isolates tested, Pseudomonas was more lethal to mealworms than Serratia or Enterococcus in both assays. Injection the bacteria at 108 cfu/ml increased their pathogenicity compared to 102-106 cfu/ml regardless of the bacteria species. The association between the entomotoxic bacteria and the nematode confirms the entomopathogenic nature of these isolates of Oscheius from Hawaii. The implication of the findings from this study showed that Oscheius indigenous to Hawaii can offer effective biological control against some insects without having to go through importation regulation and would be a good alternatives to conventional insecticides that are harmful to the environment.
Evaluating Effects of No-Till Cover Cropping Systems on Indigenous Entomopathogenic Nematodes and Fungi
(University of Hawaii at Manoa, 2017-12) Marquez, Josiah M. K.; Tropical Plant Pathology
Entomopathogens, entomopathogenic nematodes (EPN) and entomopathogenic fungi (EPF), are promising biocontrol agents for insect pest management. Use of entomopathens in Hawaii is challenged by quarantine restrictions and the failure of introduced entomopathens to persist in the field. This research focused on enhancing indigenous EPN (unknown Heterorhabditis sp.) populations in the field through conservation agriculture. We hypothesized that cover cropping with black oats (Avena strigose) or oil radish (Raphanus sativus ssp. oleiferus) followed by a no-till practice in an Oxisol soil would provide a favorable environment for EPN via provision of an organic mulch, reduced soil disturbance, improved water conservation, attracting alternative insect host and possibly production of herbivore-induced volatiles (HIPVs) from cover crops. Two field experiments, Oil Radish (OR) Experiment and Black Oat (BO) Experiment, both repeated once, were conducted to compare pre-plant treatments of 1) black oat (BO) or oil radish (OR) as cover crops in no-till plots, 2) bare ground (BG) followed by conventional tillage, and 3) conventional tillage followed by soil solarization (SOL) on abundance and infectivity of EPNs using mealworm larva (Tenebrio molitor) as a bait. Data were taken every other week throughout the 3 months of corn (Zea mays) growth following the preplant treatments. Indigenous EPNs recovered were identified by sequencing the ITS region and matched undescribed species of Heterorhabitis in NCBI, labeled as H1, SGgj, and SGmg3. Soil in BO and OR had higher volumetric soil moisture, field capacity, and soil organic matter than BG and SOL (P ≤ 0.05) in all four trials. In addition, the nematode soil food web at termination of the cover crop, and monthly during corn growth had greater % fungivore (F), fungivore to bacterivore (B) plus F ratio (F/F+B), and channel index (P ≤ 0.05) than BG and SOL in Trial I, and higher % omnivores and structure index (P ≤ 0.05) than BG and SOL in Trial II throughout the corn growing season suggested that no-till with black oat residues was in favor for fungal dominated decomposition path ways in Trial I but lead to more structured soil food web in Trial II. None-the-less, EPN infectivity was greater (P ≤ 0.05) in BO than in BG and SOL based on field cage assays in both trials. BO may have provided a habitat that enhances EPN infection. Both multivariate canonical analysis conducted for data from Trial I and Trial II suggested that higher EPN infectivity was associated with soil physical properties related to water conservation and improved soil health conditions (higher EI and SI). In the Oil Radish Experiment, OR increased abundance of Heterorhabditis sp. infective juveniles (IJs) as the age of OR increased suggested that OR provided a favorable environment for EPN reproduction. Oil radish growth consistently resulted in higher EPN abundance in OR than in BG and SOL (P ≤ 0.05) at cover crop termination. However, no-till cover cropping with OR reduce EPNs abundance over time. This could be due to fast decomposition of OR residue that resulted in lack of soil coverage and unfavorable edaphic factors for EPNs. In addition, reduced abundance of herbivorous nematodes and higher occurrence of Metarhizium anisopliae from the larva bait assay in OR plots also suggested that allelopathic compounds released from oil radish (i.e. isothiocyanates) could have biofumigant effects against EPN infectivities. In conclusion, this thesis project suggested that BO cover crop in a no-till system provided favorable edaphic factors for EPN infectivity despite lack of ability to enhance IJs abundance of theses nematodes. On the other hand, OR cover cropping enhanced abundance of EPNs, but did not improve EPNs infectivity. Occurrence of M. anisopliae on larva bait assay suggested no obvious effect of no-till cover cropping with black oats, and a negative effect of oil radish cover cropping on infectivity of EPN. Future research is needed to understand if herbivore induced plant volatiles (HIPVs) are involved in increasing ability of OR to attract EPNs, and if a mixture of OR and BO cover cropping can improve EPN activities in agroecosystems.
Plant-Parasitic Nematodes Associated with Breadfruit, Artocarpus altilis (Parkinson) Fosberg
(University of Hawaii at Manoa, 2017-12) Lau, Teri J.-W.; Tropical Plant Pathology
A survey of plant-parasitic nematodes associated with breadfruit in Hawai‘i was conducted on 25 sites with breadfruit trees. Soil and breadfruit roots yielded seven genera of plant-parasitic nematodes (Helicotylenchus, Meloidogyne, Mesocriconema, Paratylenchus, Pratylenchus, Rotylenchulus, Tylenchorhynchus) and one Heteroderid. Morphometric and molecular techniques identified a root-lesion nematode found parasitizing breadfruit as Pratylenchus coffeae. Host ranges and pathogenicity of Meloidogyne javanica, Pratylenchus coffeae, and Rotylenchulus reniformis were tested on breadfruit cultivar ‘Ma‘afala’ in two experiments. In experiment 1, P. coffeae reproduced 17-fold, while M. javanica and R. reniformis did not replace the inoculum concentration. At termination, no visible symptoms were present on host plants, but a greater root weight of breadfruit inoculated with P. coffeae was measured. Experiment 2 showed similar trends despite higher greenhouse temperatures and pot-bound breadfruit trees. Reproductive factor results indicate breadfruit is a good host to P. coffeae and a poor host to M. javanica and R. reniformis. Key Words: Hawaii, Helicotylenchus dihystera, host range, identification, Kauaʻi, Ma‘afala, Maui, Meloidogyne javanica, lesion, Ma‘afala, molecular, morphometric, morphology, Oʻahu, Paratylenchus, pathogenicity test, Pratylenchus coffeae, Rotylenchulus reniformis, survey, ‘ulu.
Functional Characterization of Putative Effector Genes of Basil Downy Mildew Pathogen Peronospora belbahrii
(University of Hawaii at Manoa, 2017-05) Shao, Dandan; Tropical Plant Pathology
Peronospora belbahrii, the causal agent of the devastating downy mildew disease on basil, is an obligate biotrophic oomycete. Similar to other oomycete pathogens, P. belbahrii is believed to secrete effectors to facilitate host colonization. To this end, we did the functional characterization of 10 P. belbahrii effector candidate genes, which encode predicted secreted proteins with a translocation motif RXLR (or RXLR-EER) and/or nuclear localization signals (NLS). First, we determined their gene expression patterns during infection using reverse transcription quantitative PCR (RT-qPCR). Five genes were induced during infection and the functionality of their predicted signal peptides was confirmed using a yeast invertase secretion system, suggesting that these genes likely encode bona fide effectors that play significant roles in manipulating host cellular processes to cause disease. Their roles in pathogenicity are currently being tested through overexpression and host-induced gene silencing (HIGS). To facilitate the genetic analysis of these and additional effector candidate genes, we developed a transient expression system in basil for utilizing overexpression and HIGS in a transient manner. To better analyze the effect resulted from the in planta expression of overexpression and HIGS construct of PbEC2 on pathogen growth, a quantitative PCR approach was developed to quantify the pathogen biomass. In addition, we generated transgenic basil to express double-stranded RNAs of one selected effector gene to determine its function in pathogenicity using HIGS. This study is expected to facilitate the understanding of P. belbahrii pathogenesis and help develop tools to control this pathogen.
Molecular Characterization of Ti Ringspot Associated Emara Virus and the Development of Assays for Its Detection
(University of Hawaii at Manoa, 2016-12) Park, Adam
Ti ringspot disease (TRD) is an emerging virus-like disease of ti plants (Cordyline fruticosa L.) spreading throughout the Hawaiian Islands. Foliar symptoms include chlorotic lesions constricted by secondary veins or circular ring-spots that can coalesce into amorphous lesions. A degenerate reverse transcription (RT)-PCR assay targeting the polymerase gene of known emaraviruses amplified a product from the RNA of symptomatic but not asymptomatic ti plants. The RT-PCR products were sequenced and found to represent a new virus species, designated ti ringspot associated virus (TiRaV). Next generation sequencing of a double-stranded RNA library provided additional sequence information on TiRaV, allowing the development of detection assays able to reliably detect TiRaV and further associate it with TRD. Phylogenetic analyses of this sequence data revealed that TiRaV forms a clade with the emaraviruses Raspberry leaf blotch virus and High Plains wheat mosaic virus in the unassigned plant virus genus Emaravirus.
Genetic Diversity, Development of Improved Diagnostic Assays and Evidence towards the Taro Planthopper (Tarophagus proserpina) as the Vector for Taro Vein Chlorosis Virus (TAVCV) in Hawaii
(University of Hawaii at Manoa, 2016-12) Gosai, Riten
Taro vein chlorosis virus (TaVCV; genus Nucleorhabdovirus, family Rhabdoviridae) is a recent discovery in Hawaii and causes veinal chlorosis with a netted appearance, stunting and petiole streaking in taro (Colocasia esculenta). Plant death may occur in severe infections. Nucleotide and amino acid sequence comparisons and phylogenetic analyses revealed extremely low levels of genetic diversity in the partial RNA-dependent RNA polymerase (RdRp) gene of 43 Hawaiian and 3 Palauan TaVCV isolates. This sequence information was used to design six new primer pairs targeting different regions of the RdRp gene. Primer set DCGF5/DCGR5 was identified as the most efficient of the six. Following optimization, highly sensitive and robust reverse transcription-polymerase chain reaction (RT-PCR) and immunocapture-RT-PCR (IC-RT-PCR) assays were developed. Localization of TaVCV in insect body parts essential for propagative, circulative virus transmission suggest that the taro planthopper, Tarophagus proserpina, is a vector of TaVCV.
Mentha spicata - A Potential Cover Crop for Tropical Conservation Agriculture
(University of Hawaii at Manoa, 2016-12) Chan, Kevin
Mint (Mentha sp.) is an important crop used raw or processed into oil for a variety of consumption purposes. This thesis evaluated mint for its potential to be a valuable living mulch in conservation agriculture-vegetable production. Furthermore, mint, as a living mulch, creates multiple benefits including reduction of soil erosion, reduction of water and nutrient runoff, retention of water, and reduction of weeds and pests. Mint susceptibility to plant-parasitic nematodes and the profitability of mint in a vegetable agro ecosystem have not been well studied. A greenhouse experiment was conducted to examine susceptibility of spearmint (Mentha spicata) and peppermint (Mentha pepreti) to root-knot (Meloidogyne incognita, M. javanica) and reniform (Rotylenchulus. reniformis) nemtodoes. Both mint species were not a host to M. incognita, M. javanica, nor R. reniformis. The nematode reproductive factors (Rf = final nematode population/initial nematodes population) after 2 months were all < 1.0 for these nematodes on mint. A micro-plot field experiment was conducted to examine the effect of spearmint living mulch eggplant vegetable system compared to a bare ground eggplant vegetable system. The spearmint living mulch did not affect eggplant yield throughout the 14 months of the experiment as compared to the bare ground system. After the initial year of establishment, additional profit from harvesting spearmint for sale was estimated to be $20,949.06 per hectare annually based on a partial budget economic analysis. Planting spearmint as a living mulch in an eggplant agro ecosystem provides an economically viable conservation agricultural production system in a water resourceful environment.

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