Browsing by Author "Roy, Kylle"
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Item A rapid diagnostics test and mobile "Lab-in-a-suitcase" platform for detecting Ceratocystis spp. responsible for rapid `Ōhi`a death(2017-04-23) Atkinson, Carter T.; Watcher-Weatherwax, William; Roy, Kylle; Heller, Wade P.; Keith, Lisa M.We describe a field compatible molecular diagnostic test for two new species of Ceratocystis that infect `ōhi`a (Metrosideros polymorpha) and cause the disease commonly known as Rapid `Ōhi`a Death. The diagnostic is based on amplification of a DNA locus within the internal transcribed spacer region that separates fungal 5.8S ribosomal genes. The assay uses forward and reverse primers, recombinase polymerase, and a fluorescent probe that allows isothermal (40oC) amplification and simultaneous quantification of a 115 base pair product with a battery operated fluorometer. DNA extractions are field compatible and can be done by heating wood drill shavings to 100oC in Instagene® solution containing Chelex® resin to bind potential amplification inhibitors. The initial heat treatment is followed by a short bead beating step with steel ball bearings and zirconium beads to release DNA. DNA is subsequently purified with a magnetic bead based extraction method that does not require silica columns or centrifugation. The assay is designed around a portable “lab-in-a-suitcase” platform that includes a portable fluorometer, miniature centrifuge, and heat block that operate off either 120V AC power sources or a 12 volt battery with a portable inverter, a magnetic rack designed for 1.5 ml tubes and magnetic bead DNA purification, pipettes and consumable reagents and tubes. The entire assay from DNA extraction to results can be performed in less than 90 minutes on up to six independent samples plus a positive and negative control. Sensitivity based on suspensions of Ceratocystis endoconidia (spores) that were added to wood shavings and processed under field conditions by Instagene® magnetic bead DNA extraction was up to 163 spores/mg wood for Species A and 55 spores/mg wood for Species B in 95% of replicates as determined by probit analysis. Sensitivity increased 5–10 fold to 19 spores/mg wood for Species A and 9 spores/mg wood for Species B when extractions were performed with a commercial, silica column based DNA purification kit. The test did not cross react with other common fungi that have been isolated from `ōhi`a.Item Economical environmental sampler designs for detecting airborne spread of fungi responsible for Rapid `Ōhi`a Death(2019-02-10) Atkinson, Carter T.; Roy, Kylle; Granthon, CarolinaWe designed two new samplers for monitoring airborne particulates that rely on either natural wind currents (Passive Environmental Sampler) or a battery-operated fan (Active Environmental Sampler). Both samplers are significantly less expensive than commercial devices such as Rotorod® and Burkard Samplers that are used in the agricultural and health science industries. They are economical enough to be deployed in large numbers across broad landscapes. We evaluated their use for detecting airborne spread of ambrosia beetle frass that may contain infective spores of the fungi (Ceratocystis lukuohia and C. huliohia) that are responsible for Rapid `Ōhi`a Death (ROD), a newly documented pathosystem on Hawai`i Island. We compared performance of the new samplers to Rotorod® Model 20 Samplers by releasing synthetic polyethylene spheres (12–160 µm in diameter) and also Xyleborus spp. frass known to contain C. lukuohia and C. huliohia propagules under controlled laboratory and field conditions. Overall, the Active Environmental Sampler proved to be 3–4 times more effective in capturing polyethylene spheres and 2–3 times more effective in capturing frass than either the Passive or Rotorod® Samplers. Significant differences between the Passive and Rotorod® Samplers were not detected. For the frass release experiment, C. lukuohia DNA was detected once by qPCR in an Active Environmental Sampler and C. huliohia DNA was detected during two different trials, once with an Active Environmental Sampler and once with a Passive Environmental Sampler. No detections were made with Rotorod® Samplers. Both Active and Passive Samplers were used in the field for detection of airborne dispersal of C. lukuohia and C. huliohia at Orchidlands Estates in the Puna District of Hawai`i Island. We found that airborne dispersal of potentially infective beetle frass was uncommon over short distances with qPCR detections in up to 10% of weekly sampler collections.Item Effectiveness of rapid 'ōhi'a death management strategies at a focal disease outbreak on Hawai'i Island(2021-02-03) Roy, Kylle; Granthon, Carolina; Peck, Robert W.; Atkinson, Carter T.The ongoing spread of rapid ‘ōhi‘a death (ROD) in the Hawaiian Islands threatens the long-term sustainability of ‘ōhi‘a lehua (Metrosideros polymorpha) forests throughout the state. First identified in the Puna district of Hawai‘i Island in 2014, the disease caused by the novel fungi Ceratocystis lukuohia and Ceratocystis huliohia has now spread island-wide and was recently detected on Kaua‘i, O‘ahu, and Maui. The leading hypothesis for the spread of ROD is through airborne ambrosia beetle frass particles that contain viable Ceratocystis propagules, thus management efforts focus on containing this frass. At the time of this study (2017–2018), the Waipunalei site was the northernmost outbreak of ROD on Hawai‘i Island. The focal nature of the outbreak and accessibility of the location provided the opportunity to monitor the effectiveness of two types of proposed management methods to reduce the airborne spread of potentially infective ambrosia beetle frass: tree felling and insecticide treatments. We placed 23 passive environmental samplers (PES), which monitored for airborne frass and wood particles containing C. lukuohia and C. huliohia in a grid that spanned the outbreak area over 22 weeks. Cross-vane panel traps with 1:1 methanol:ethanol lures were attached to nine of the PES to document wood-boring ambrosia and cerambycid beetle populations during the latter three months of the study. Monitoring with PES began three weeks before management and continued for one month after the last infected trees were felled. Glass microscope slides from the 23 PES were examined for airborne ambrosia beetle frass and wood particles by microscopy. DNA was extracted from the slides and tested by qPCR (quantitative polymerase chain reaction) for C. lukuohia and C. huliohia. We also investigated the correlation of beetle gallery counts with tree height and tested the efficacy of Bifen I/T insecticide (active ingredient: bifenthrin 7.9%) for preventing beetle attacks on the cut surface of ‘ōhi‘a bolts (tree stem sections). Beetle trapping data revealed that the area supports a diverse community of wood-boring beetles, some of which likely attack ‘ōhi‘a and may facilitate the spread of ROD. The number of beetle galleries on felled ‘ōhi‘a trees decreased linearly as tree height increased. We also observed significantly fewer beetle attacks on Bifen I/T treated ‘ōhi‘a bolts than non-treated bolts, but gallery formation nearly ceased in both treated and control bolts by week three. Ceratocystis lukuohia DNA was detected twenty-six times and C. huliohia was detected five times in the PES throughout this study. DNA detections were correlated to frass and wood counts, and the number of felled trees were correlated to wood particle counts but not frass counts. Both the timing and distribution of detections across the sampling grid indicate that tree felling may have reduced airborne detections of Ceratocystis DNA soon after tree felling was completed. A subsequent increase in detections after tree felling ceased may indicate that incomplete removal of infected trees and the appearance of new infections in previously asymptomatic trees could have allowed airborne detections of potentially infectious fungal propagules to once again increase.Item Food Web Analysis of Hawai‘i Island’s Blackburnia hawaiiensis (Coleoptera: Carabidae) Using Next-generation Sequencing and Stable Isotope Techniques(2015) Roy, Kylle; Price, Donald K.; Tropical Conservation Biology & Environmental ScienceTropical montane forests are valuable ecosystems in Hawai‘i, providing fresh water to the people of the islands as well as acting as reservoirs of biodiversity. These forests are experiencing rapid alterations due to anthropogenic effects such as climate change, habitat degradation, invasive species, and industrialization. Some of the detrimental effects caused by these ecosystem alterations can be mitigated through understanding the genetics and ecology of the organisms within it. Despite the importance of these arthropod-dominated ecosystems, knowledge of food webs and predator-prey interactions is sparse. In order to supplement the understanding of Hawai‘i’s montane forest ecosystems, we have implemented two different methods of diet analysis on the endemic Hawaiian carabid beetle, Blackburnia hawaiiensis. This understudied carabid may provide important ecosystem functions, being a numerically dominant predatory insect and widely distributed throughout Hawai‘i Island. B. hawaiiensis populations and potential prey in similar, highly isolated geographic locations were used to employ two different yet complimentary laboratory techniques: natural abundance stable isotope analysis (SIA) and metagenomics of gut contents using next-generation sequencing (NGS). Both NGS and SIA have revealed B. hawaiiensis to be a high trophic consumer with evidence of intraguild predation in three study sites: Ka‘iholena, Thurston, and Pu‘u Maka‘ala. In a broader context, the combined SIA and NGS techniques have great potential to further our understanding of the arthropod food webs of the montane forests of Hawai‘i Island, ultimately improving conservation efforts for the entire arthropod community. These two methods in combination could potentially be implemented in any ecosystem globally to better determine the diets of species within complex food webs, enhancing ecosystem management strategies.