M.S. - Tropical Plant Pathology

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    Performance of conventional and biological fungicides for the management of coffee leaf rust and coffee anthracnose
    (2023) Buchholz, Elizabeth Rose; Cheng, Zhiqiang; Tropical Plant Pathology
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    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.
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    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.
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    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.
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    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.
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    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.
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    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.