Scholarly Works, Virginia Agricultural Experiment Station

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https://hdl.handle.net/10919/24309

VAES faculty are located at 11 Agricultural Research and Extension Centers in Virginia and three colleges at Virginia Tech (CALS, CNRE, and VMRCVM).

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Assessing Economic Contributions of the Virginia Seafood Industry: An Estimation Framework Utilizing Primary Data
Gonçalves, Fernando H.; van Senten, Jonathan; Schwarz, Michael H.; Hegde, Shraddha (MDPI, 2024-05-26)
With a focus on seafood industries, this study provides a framework for economic contribution assessments, outlines Virginia’s seafood supply chain components, and evaluates the direct, indirect, and induced economic impacts of Virginia’s seafood industry in 2019. Utilizing an analysis-by-parts method in IMPLAN, primary expenditure data from watermen, aquaculture farmers, processors, and distributors were collected through surveys. The efficacy of obtaining primary data through stakeholder surveys heavily relies on the investigator’s interpersonal skills to establish trust and elucidate the study’s benefits, particularly its potential to inform policy decisions. In 2019, the Virginia seafood industry’s estimated total economic contributions amounted to USD 1.1 billion, supporting 7187 individuals. This impact encompasses 6050 direct jobs, 523 indirect jobs, and 614 induced jobs, primarily benefiting watermen and coastal communities. Furthermore, the industry’s influence extends beyond its immediate economic sphere, supporting diverse sectors such as polystyrene foam manufacturing, boat building, sporting and athletic goods, and commercial and industrial machinery. Wages and salaries disbursed throughout the seafood supply chain ripple to Virginia’s economy, benefiting nondepository credit intermediation, owner-occupied dwellings, and real estate sectors. Future research focusing on seafood sales in restaurants and retail outlets will complete the understanding of the seafood industry’s broader economic impact on the state.
Phenotypic Dissection of Drought Tolerance in Virginia and Carolinas within a Recombinant Inbred Line Population Involving a Spanish and a Virginia-Type Peanut Lines
Kumar, Naveen; Haak, David C.; Dunne, Jeffrey C.; Balota, Maria (MDPI, 2024-06-08)
Peanut (Arachis hypogaea L.) is a rainfed crop grown in both tropical and subtropical agro-climatic regions of the world where drought causes around 20% yield losses per year. In the United States, annual losses caused by drought are around $50 million. The objective of this research was to (1) identify genetic variation for the normalized difference vegetation index (NDVI), canopy temperature depression (CTD), relative chlorophyll content by SPAD reading (SCMR), CO₂ assimilation rate, and wilting among recombinant inbred lines (RILs) derived from two diverse parents N08086olJCT and ICGV 86015, to (2) determine if the physiological traits can be used for expediting selection for drought tolerance, and (3) experimental validation to identify lines with improved yield under water-limited conditions. Initially, 337 lines were phenotyped under rainfed production and a selected subset of 52 RILs were tested under rainout shelters, where drought was imposed for eight weeks during the midseason (July and August). We found that under induced drought, pod yield was negatively correlated with wilting and CTD, i.e., cooler canopy and high yield correlated positively with the NDVI and SPAD. These traits could be used to select genotypes with high yields under drought stress. RILs #73, #56, #60, and #31 performed better in terms of yield under both irrigated and drought conditions compared to check varieties Bailey, a popular high-yielding commercial cultivar, and GP-NC WS 17, a drought-tolerant germplasm.
Comparing Regression and Classification Models to Estimate Leaf Spot Disease in Peanut (Arachis hypogaea L.) for Implementation in Breeding Selection
Chapu, Ivan; Chandel, Abhilash; Sie, Emmanuel Kofi; Okello, David Kalule; Oteng-Frimpong, Richard; Okello, Robert Cyrus Ongom; Hoisington, David; Balota, Maria (MDPI, 2024-04-30)
Late leaf spot (LLS) is an important disease of peanut, causing global yield losses. Developing resistant varieties through breeding is crucial for yield stability, especially for smallholder farmers. However, traditional phenotyping methods used for resistance selection are laborious and subjective. Remote sensing offers an accurate, objective, and efficient alternative for phenotyping for resistance. The objectives of this study were to compare between regression and classification for breeding, and to identify the best models and indices to be used for selection. We evaluated 223 genotypes in three environments: Serere in 2020, and Nakabango and Nyankpala in 2021. Phenotypic data were collected using visual scores and two handheld sensors: a red–green–blue (RGB) camera and GreenSeeker. RGB indices derived from the images, along with the normalized difference vegetation index (NDVI), were used to model LLS resistance using statistical and machine learning methods. Both regression and classification methods were also evaluated for selection. Random Forest (RF), the artificial neural network (ANN), and k-nearest neighbors (KNNs) were the top-performing algorithms for both regression and classification. The ANN (R²: 0.81, RMSE: 22%) was the best regression algorithm, while the RF was the best classification algorithm for both binary (90%) and multiclass (78% and 73% accuracy) classification. The classification accuracy of the models decreased with the increase in classification classes. NDVI, crop senescence index (CSI), hue, and greenness index were strongly associated with LLS and useful for selection. Our study demonstrates that the integration of remote sensing and machine learning can enhance selection for LLS-resistant genotypes, aiding plant breeders in managing large populations effectively.
Evaluation of Production and Pest Management Practices in Peanut (Arachis hypogaea) in Ghana
Seidu, Ahmed; Abudulai, Mumuni; Dzomeku, Israel K.; Mahama, Georgie Y.; Nboyine, Jerry A.; Appaw, William; Akromah, Richard; Arthur, Stephen; Bolfrey-Arku, Grace; Mochiah, M. Brandford; Jordan, David L.; Brandenburg, Rick L.; MacDonald, Greg; Balota, Maria; Hoisington, David; Rhoads, Jamie (MDPI, 2024-05-06)
The economic return for peanut (Arachis hypogaea L.) in Ghana is often low due to limitations in the availability of inputs or their adoption, which are needed to optimize yield. Six experiments were conducted in Ghana in 2020 and 2021 to determine the impact of planting date, cultivar, fertilization, pest management practices, and harvest date on peanut yield, financial return, and pest reaction. A wide range of interactions among these treatment factors were often observed for infestations of aphids (Aphis gossypii Glover); groundnut rosette disease (Umbravirus: Tombusviridaee); millipedes (Peridontopyge spp.); white grubs (Schyzonicha spp.); wireworms (Conoderus spp.); termites (Microtermes and Odontotermes spp.); canopy defoliation as a result of early leaf spot disease caused by Passalora arachidicola (Hori) and late leaf spot caused by Nothopassalora personata (Berk. and M. A. Curtis); and the scarification and boring of pods caused by arthropod feeding. Pod yield and economic return increased for the cultivar Chitaochi and Sarinut 2 when fertilizer was applied and when fertilizer was applied at early, mid-, and late planting dates. Pod yield and economic return increased when a combination of locally derived potassium soaps was used for aphid suppression and one additional hand weeding was used in the improved pest management practice compared with the traditional practice without these inputs. Pearson correlations for yield and economic return were negatively correlated for all pests and damage caused by pests. The results from these experiments can be used by farmers and their advisors to develop production packages for peanut production in Ghana.
New Species-Specific Real-Time PCR Assays for Colletotrichum Species Causing Bitter Rot of Apple
McHenry, Diana J.; Aćimović, Srđan G. (MDPI, 2024-04-27)
Bitter rot of apple is an economically important worldwide disease caused by different Colletotrichum species, depending on many factors such as climate, geography, other hosts, and crop management practices. Culture, morphology, and single-locus sequencing-based methods for identifying the Colletotrichum species are severely limited in effectiveness, while the multilocus sequence typing methods available for delineating species are costly, time-intensive, and require high expertise. We developed species-specific hydrolysis probe real-time PCR assays for the following nine Colletotrichum species causing bitter rot in the Mid-Atlantic U.S.A.: C. fructicola, C. chrysophilum, C. noveboracense, C. gloeosporioides s.s., C. henanense, C. siamense and C. theobromicola from the C. gloeosporioides species complex, and C. fioriniae and C. nymphaeae from the C. acutatum species complex. After searching 14 gene regions, we designed primers and probes in 5 of them for the nine target species. Four primer–probe set pairs were able to be duplexed. Sensitivity tests showed as little as 0.5 pg DNA were detectable. These real-time PCR assays will provide rapid and reliable identification of these key Colletotrichum species and will be critically important for studies aiming to elucidate their biology, epidemiology, and management on apples as the number one produced and consumed tree fruit in the U.S.A.
Evaluating Different Methods to Establish Biodiverse Swards of Native Grasses and Wildflowers for Pasturelands
Kubesch, Jonathan O. C.; Greiner, Scott P.; Pent, Gabriel J.; Reid, J. Leighton; Tracy, Benjamin F. (MDPI, 2024-05-14)
Many cool-season pastures in the southeastern U.S. are dominated by a competitive cool-season grass, tall fescue (Schedonorus arundinaceus), and lack substantial plant diversity. Planting native warm-season grasses (NWSGs) and wildflowers (WFs) into these pastures could provide summer forage for cattle and more floral resources for pollinators. This paper summarizes field experiments designed to evaluate different spatiotemporal planting arrangements of NWSGs and WFs to improve their establishment success. The study was conducted from April 2021 to October 2023 in central Virginia (USA). Planting treatments included NWSG and WF mixtures planted: (1) together in the same space, (2) spatially separated in space (i.e., side by side), or (3) temporally separated where NWSGs and WFs were planted in difference sequences. Results showed few differences in forage mass, floral production, and botanical composition as well as stand density in 2021 and 2022. In 2023, NWSG abundance was greater where grasses were planted first or mixed with WFs. Similarly, the WF component was favored when they were planted before NWSGs. Overall, planting NWSG and WF mixes separately, either spatially or temporally, favors successful establishment and could offer more flexibility for using selective herbicides to suppress the heavy weed pressure that often accompanies these plantings.
Phenotyping Peanut Drought Stress with Aerial Remote-Sensing and Crop Index Data
Balota, Maria; Sarkar, Sayantan; Bennett, Rebecca S.; Burow, Mark D. (MDPI, 2024-04-02)
Peanut (Arachis hypogaea L.) plants respond to drought stress through changes in morpho-physiological and agronomic characteristics that breeders can use to improve the drought tolerance of this crop. Although agronomic traits, such as plant height, lateral growth, and yield, are easily measured, they may have low heritability due to environmental dependencies, including the soil type and rainfall distribution. Morpho-physiological characteristics, which may have high heritability, allow for optimal genetic gain. However, they are challenging to measure accurately at the field scale, hindering the confident selection of drought-tolerant genotypes. To this end, aerial imagery collected from unmanned aerial vehicles (UAVs) may provide confident phenotyping of drought tolerance. We selected a subset of 28 accessions from the U.S. peanut mini-core germplasm collection for in-depth evaluation under well-watered (rainfed) and water-restricted conditions in 2018 and 2019. We measured morpho-physiological and agronomic characteristics manually and estimated them from aerially collected vegetation indices. The peanut genotype and water regime significantly (p < 0.05) affected all the plant characteristics (RCC, SLA, yield, etc.). Manual and aerial measurements correlated with r values ranging from 0.02 to 0.94 (p < 0.05), but aerially estimated traits had a higher broad sense heritability (H²) than manual measurements. In particular, CO₂ assimilation, stomatal conductance, and transpiration rates were efficiently estimated (R² ranging from 0.76 to 0.86) from the vegetation indices, indicating that UAVs can be used to phenotype drought tolerance for genetic gains in peanut plants.
Lasiodiplodia iraniensis and Diaporthe spp. Are Associated with Twig Dieback and Fruit Stem-End Rot of Sweet Orange, Citrus sinensis, in Florida
Piattino, Valeria; Aiello, Dalia; Dardani, Greta; Martino, Ilaria; Flores, Mauricio; Aćimović, Srđan G.; Spadaro, Davide; Polizzi, Giancarlo; Guarnaccia, Vladimiro (MDPI, 2024-04-17)
Florida ranks among the most important citrus growing regions in the USA. The present study investigates the occurrence, diversity, and pathogenicity of fungal species associated with symptomatic sweet orange (Citrus sinensis) cv. Valencia plants and fruit. The survey was conducted on twigs and fruit collected in Southwest Florida during 2022. Based on morphological and molecular characteristics, the identified isolates belonged to the species Lasiodiplodia iraniensis, Diaporthe pseudomangiferae, and Diaporthe ueckerae. The pathogenicity of representative isolates was evaluated on citrus fruit and plants. Lasiodiplodia iraniensis was the most virulent on fruit and plants, followed by Diaporthe pseudomangiferae. Diaporthe ueckerae had the lowest virulence on fruit, and it was not pathogenic to plants. In vitro tests were performed to assess the effect of temperature on mycelial radial growth. The optimum temperature of growth ranged from 26.0 to 28.4 °C for all the evaluated species, and L. iraniensis showed the fastest mycelial growth. This study represents the first report of L. iraniensis as a causal agent of tree dieback and fruit stem-end rot on C. sinensis worldwide. Moreover, D. pseudomangiferae and D. ueckerae are reported here for the first time in association with citrus diseases worldwide.
Transcriptome Profiling of a Salt Excluder Hybrid Grapevine Rootstock ‘Ruggeri’ throughout Salinity
Gajjar, Pranavkumar; Ismail, Ahmed; Islam, Tabibul; Moniruzzaman, Md; Darwish, Ahmed G.; Dawood, Ahmed S.; Mohamed, Ahmed G.; Haikal, Amr M.; El-Saady, Abdelkareem M.; El-Kereamy, Ashraf; Sherif, Sherif M.; Abazinge, Michael D.; Kambiranda, Devaiah; El-Sharkawy, Islam (MDPI, 2024-03-14)
Salinity is one of the substantial threats to plant productivity and could be escorted by other stresses such as heat and drought. It impairs critical biological processes, such as photosynthesis, energy, and water/nutrient acquisition, ultimately leading to cell death when stress intensity becomes uncured. Therefore, plants deploy several proper processes to overcome such hostile circumstances. Grapevine is one of the most important crops worldwide that is relatively salt-tolerant and preferentially cultivated in hot and semi-arid areas. One of the most applicable strategies for sustainable viticulture is using salt-tolerant rootstock such as Ruggeri (RUG). The rootstock showed efficient capacity of photosynthesis, ROS detoxification, and carbohydrate accumulation under salinity. The current study utilized the transcriptome profiling approach to identify the molecular events of RUG throughout a regime of salt stress followed by a recovery procedure. The data showed progressive changes in the transcriptome profiling throughout salinity, underpinning the involvement of a large number of genes in transcriptional reprogramming during stress. Our results established a considerable enrichment of the biological process GO-terms related to salinity adaptation, such as signaling, hormones, photosynthesis, carbohydrates, and ROS homeostasis. Among the battery of molecular/cellular responses launched upon salinity, ROS homeostasis plays the central role of salt adaptation.
Effects of Grosmannia clavigera and Leptographium longiclavatum on Western White Pine Seedlings and the Fungicidal Activity of Alamo®, Arbotect®, and TREE-äge®
Wyka, Stephen A.; Doccola, Joseph J.; Strom, Brian L.; Smith, Sheri L.; McPherson, Douglas W.; Aćimović, Srđan G.; Klepzig, Kier D. (International Society of Arboriculture, 2016-03-15)
Bark beetles carry a number of associated organisms that are transferred to the host tree upon attack that are thought to play a role in tree decline. To assess the pathogenicity to western white pine (WWP; Pinus monticola) of fungi carried by the mountain pine beetle (MPB; Dendroctonus ponderosae), and to evaluate the potential for systemic prophylactic treatments for reducing fungal impacts, experiments were conducted with WWP seedlings to meet three objectives: 1) evaluate pathogenicity of two MPB-associated blue-stain fungi; 2) evaluate phytotoxicity of tree injection products; 3) evaluate the anti-fungal activity of tree injection products, in vitro and in vivo, toward the associated blue-staining fungi. To evaluate pathogenicity, seedlings were inoculated with Grosmannia clavigera or Leptographium longiclavatum, common fungal associates of MPB. Seedling mortality at four months after inoculation was 50% with L. longiclavatum and 90% with G. clavigera, both significantly higher than controls and thereby demonstrating pathogenicity. Phytotoxic effects of TREE-äge®, Alamo®, and Arbotect® were evaluated by stem injection; no phytotoxic effects were observed. Anti-fungal properties of the same three products were evaluated in vitro against G. clavigera, where Alamo was most active. Co-inoculation of G. clavigera and L. longiclavatum into seedlings after a stem injection of Alamo showed significantly less mortality and lesion formation than either species alone. Results support the hypothesis that MPB blue-stain associates, particularly G. clavigera, promote death of WWP when attacked by MPB. These findings suggest that the administration of a fungicide with insecticide for tree protection against bark beetles may be advantageous.
Eutypa Dieback and ESCA - Trunk Necrosis and Dieback Diseases of Grapevines in Serbia
Aćimović, Srđan G.; Delibašić, Goran; Schilder, Annemiek; Tanovic, Brankica (University of Novi Sad, Serbia, 2008-12-22)
Evaluation of Trunk-injected Bactericides and Prohexadione-calcium for Environmentally Friendly Control of Fire Blight (Erwinia amylovora) in Apples
Aćimović, Srđan G.; McGhee, Gayle C.; Sundin, George W.; Wise, John C. (Plant Protection Society of Serbia (PPSS), 2015-11-18)
Trunk injection is a novel delivery method for plant protective compounds in agriculture. It could increase sustainability of fruit production through target-precise disease control. We evaluated trunk-injected antibiotics, copper chelate (CC), and prohexadione-calcium (PC) in control of fire blight on apples. After 1-2 spring injections of oxytetracycline (OX), kasugamycin (KS) and CC, we evaluated inoculated apple trees for blossom and shoot blight incidence. In a separate study, after spraying or injection of PC, we evaluated shoot blight severity after inoculation. At a high disease pressure, OX, KS and CC provided blossom blight control of 60.6, 31.7 and 15.5-17.8%, respectively. The same compounds provided control of shoot blight incidence of 60.7, 42 and 24.5-33.9%, respectively. The results indicate that shoots initially accumulate more of the injected compound than flowers, due to their higher transpiration driven by larger green tissue area. Sprayed PC reduced shoot blight severity for 25.6% and caused expected reduction of shoot length, while trunk-injected PC failed to cause any of these effects. This indicates that PC did not translocate into the canopy due to its strong binding in the xylem. With the development and use of injectable formulations, proper dosing, and optimal injection timing, delivered compounds could have more time for accumulation in the canopy and thus provide better fire blight control. Hence, trunk injection could become an effective option for fire blight control on apple trees.
Some important fungal pathogens of medicinal plants in Južna-Bačka region
Balaž, Jelica S.; Aćimović, Srđan G.; Crnobarac, Jovan; Adamović, Dušan; Jacimovic, Goran (University of Novi Sad, Serbia, 2010-01-01)
Erwinia amylovora in Vojvodina and Measures of Control
Balaž, Jelica S.; Keserović, Zoran; Aćimović, Srđan G.; Nikolic, Zorica; Mažić, Jelena (University of Novi Sad, Serbia, 2009-02-10)
Bacterioses of Soybean
Balaž, Jelica S.; Aćimović, Srđan G. (University of Novi Sad, Serbia, 2008-10-01)
Cultural characteristics of Eutypa lata and one unknown isolate on different nutrient media
Aćimović, Srđan G.; Delibašić, Goran (University of Kragujevac, Serbia, 2005-08)
Proof of concept for management of shoot blight and fire blight cankers on pear with preventive spray applications of giant knotweed extract
Borba, Matheus C.; Meredith, Christopher L.; Dhar, Bidhan C.; Aćimović, Srđan G. (Frontiers, 2023-03-16)
With the goal to develop management tools to mitigate the severity of shoot blight on pear caused by Erwinia amylovora and prevent fire blight canker development from infected shoots on pear tree wood, we evaluated five preventive treatments in inoculated experiments over 2-years. We focused on foliar spray and trunk injection applications of extract of giant knotweed, Reynoutria sachalinensis (RSE), as a plant resistance activator candidate and on comparisons to antibiotics. Five foliar applications of RSE (2.24 L/ha) on 6-year-old trees of cultivar ‘Bartlett’ resulted in 100% control of shoot blight severity and canker incidence in both years of evaluation. The RSE trunk injection in fall of the first year did not control the disease allowing 75% of shoot severity and 38.5% of canker incidence. The opposite happened in the second year, when we injected RSE in spring, providing 85.7% control of shoot blight severity and 89.8% control of canker incidence. Antibiotics oxytetracycline (OTC) and streptomycin (STM) did not provide consistent control in the second year in comparison to the first year. In the first year, both antibiotics, OTC (trunk injection and foliar spray) and STM were effective, providing 94.8%, 100% and 86.3% control of shoot blight, respectively. They also provided 100, 91 and 84% control of canker incidence, respectively. However, in the second they were not effective allowing 69 – 96% of shoot blight severity and 70 – 92% of canker incidence. Regardless of the inconsistency in the effectiveness of antibiotics, which are not recommended for shoot blight control due to risks of promoting antibiotic resistance, five preventive spray applications of RSE could be used on pear trees to prevent shoot blight and canker development. Thus, the RSE spray program we developed could serve as a valuable control tool that could help prevent sudden outbreaks of shoot blight during summer and pear tree death from fire blight cankers with no risk for pathogen resistance.
Genomic Resources of Four Colletotrichum Species (C. fioriniae, C. chrysophilum, C. noveboracense, and C. nupharicola) Threatening Commercial Apple Production in the Eastern United States
Khodadadi, Fatemeh; Giroux, Emily; Bilodeau, Guillaume J.; Jurick, Wayne M. II; Aćimović, Srđan G. (American Phytopathological Society, 2023-03-07)
The genus Colletotrichum includes nine major clades with 252 species and 15 major phylogenetic lineages, also known as species complexes. Colletotrichum spp. are one of the top fungal plant pathogens causing anthracnose and pre- and postharvest fruit rots worldwide. Apple orchards are imperiled by devastating losses from apple bitter rot, ranging from 24 to 98%, which is a serious disease caused by several Colletotrichum species. Bitter rot is also a major postharvest rot disease, with C. fioriniae causing from 2 to 14% of unmarketable fruit in commercial apple storages. Dominant species causing apple bitter rot in the Mid- Atlantic United States are C. fioriniae from the Colletotrichum acutatum species complex and C. chrysophilum and C. noveboracense from the C. gloeosporioides species complex (CGSC). C. fioriniae is the dominant species causing apple bitter rot in the Northeastern and Mid-Atlantic states. C. chrysophilum was first identified on banana and cashew but has been recently found as the second most dominant species causing apple bitter rot in the Mid-Atlantic. As the third most dominant pathogen, C. noveboracense MB 836581 was identified as a novel species in the CGSC, causing apple bitter rot in the Mid-Atlantic. C. nupharicola is a sister group to C. fructicola and C. noveboracense, also causing bitter rot on apple. We deliver the resources of 10 new genomes, including two isolates of C. fioriniae, three isolates of C. chrysophilum, three isolates of C. noveboracense, and two isolates of C. nupharicola collected from apple fruit, yellow waterlily, and Juglans nigra.
PATHMAP (Pathogen And Tree fruit Health MAP): A Smartphone App and Interactive Dashboard to Record and Map Tree Fruit Diseases, Disorders, and Insect Pests
Jurick, Wayne M.; Messinger, Lindsey; Wallis, Anna; Peter, Kari A.; Villani, Sara; Bradshaw, Michael J.; Bartholomew, Holly P.; Buser, Michael; Aćimović, Srđan G.; Fonseca, Jorge M.; Cox, Kerik D. (Scientific Societies, 2022-12)
PATHMAP (Pathogen And Tree fruit Health MAP) is a smartphone application (app) and interactive dashboard developed specifically for support specialists, extension personnel, and university scientists supporting the tree fruit industry. The PATHMAP app collects detailed information about observed diseases, insect pests, and disorders and provides the option to attach photos. The data are then visualized using a graphical interface dashboard displaying an interactive color-coded map. Prior to the development of PATHMAP, abundant tree fruit disorder data were collected each year, but a central interactive repository for archiving data and facilitating communication of field observations did not exist. PATHMAP has been beta tested by university extension personnel, private consultants, and university scientists to ensure usability and functionality. PATHMAP will be used within the tree fruit industry for monitoring known pest patterns, occurrences, and outbreaks of emerging pathogens. It will augment existing extension diagnosis listservs that have value in visual diagnosis but are cumbersome and have no archiving capabilities. Data obtained through the tool can be used in epidemiological meta-analyses and to develop new predictive models, and can serve as a platform to track emerging pathogens, insects, and disorders for a variety of cropping systems. [Formula: see text] The author(s) have dedicated the work to the public domain under the Creative Commons CC0 “No Rights Reserved” license by waiving all of his or her rights to the work worldwide under copyright law, including all related and neighboring rights, to the extent allowed by law, 2022.
Improved Canker Processing and Viability Droplet Digital PCR Allow Detection of Erwinia amylovora Viable Nonculturable Cells in Apple Bark
Dhar, Bidhan Chandra; Delgado Santander, Ricardo; Aćimović, Srđan G. (MDPI, 2024-02-12)
The bacterium Erwinia amylovora causes fire blight and continues to threaten global commercial apple and pear production. Conventional microbiology techniques cannot accurately determine the presence of live pathogen cells in fire blight cankers. Several factors may prevent E. amylovora from growing on solid culture media, including competing microbiota and the release of bacterial-growth-inhibitory compounds by plant material during sample processing. We previously developed a canker processing methodology and a chip-based viability digital PCR (v-dPCR) assay using propidium monoazide (PMA) to bypass these obstacles. However, sample analysis was still time-consuming and physically demanding. In this work, we improved the previous protocol using an automatic tissue homogenizer and transferred the chip-based v-dPCR to the BioRad QX200 droplet dPCR (ddPCR) platform. The improved sample processing method allowed the simultaneous, fast, and effortless processing of up to six samples. Moreover, the transferred v-ddPCR protocol was compatible with the same PMA treatment and showed a similar dynamic range, from 7.2 × 10² to 7.6 × 10⁷ cells mL⁻¹, as the previous v-dPCR. Finally, the improved protocol allowed, for the first time, the detection of E. amylovora viable but nonculturable (VBNC) cells in cankers and bark tissues surrounding cankers. Our v-ddPCR assay will enable new ways to evaluate resistant pome fruit tree germplasm, further dissect the E. amylovora life cycle, and elucidate E. amylovora physiology, epidemiology, and new options for canker management.

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