@article{rogers_koehler_2021, title={Nondestructive Sampling to Monitor Macrophomina phaseolina Root Colonization in Overwintering Stevia}, volume={22}, ISSN={["1535-1025"]}, DOI={10.1094/PHP-10-20-0092-BR}, abstractNote={ Macrophomina phaseolina is a soilborne fungal pathogen in the family Botryosphaeriaceae. Microsclerotia of M. phaseolina were first observed at the base of overwintering stevia stems in North Carolina in spring 2016. Previous studies utilized destructive sampling methods to monitor M. phaseolina in stevia fields; however, these methods are not feasible for long-term monitoring of disease in a perennial system. In the current study, nondestructive root soil-core sampling was conducted during overwintering months, from October 2018 to January 2020, to monitor M. phaseolina root colonization in stevia in Rocky Mount, NC. Two-inch-diameter soil cores were collected through the root zone, and fresh weight of roots was recorded for each soil core. M. phaseolina recovery was evaluated by examining mycelial growth from roots plated onto potato dextrose agar. There was no significant effect of sample weight on M. phaseolina across all dates, but there was one date for which sample weight had a significant effect on recovery (P = 0.01; α = 0.05). For both recovery and sample weight, sampling date was a significant predictor (P = 1.68e-5 and P = 0.0389, respectively; α = 0.05). Weather and climate data revealed that dates with no M. phaseolina recovery had lowest mean air and soil temperatures and the greatest number of days below freezing in the month prior to sampling. In separate sampling years, October sampling dates had the highest recovery of M. phaseolina. Future field trials should determine if October samplings can predict survival and vigor of reemerging stevia plants. }, number={2}, journal={PLANT HEALTH PROGRESS}, author={Rogers, Layne W. and Koehler, Alyssa M.}, year={2021}, pages={151–153} }
 @article{koehler_shew_2019, title={Effects of fungicide applications on root-infecting microorganisms and overwintering survival of perennial stevia}, volume={120}, ISSN={0261-2194}, url={http://dx.doi.org/10.1016/J.CROPRO.2019.02.010}, DOI={10.1016/j.cropro.2019.02.010}, abstractNote={Koehler, A.M. and Shew, H.D. 2018. Effects of fungicide applications on root-infecting microorganisms and overwintering survival of perennial stevia. Crop Protection 117:000–000. Stevia (Stevia rebaudiana) is a perennial species emerging as a new crop in the southeastern United States. In previous studies, application of quinone outside inhibitor (QoI) fungicides enhanced overwintering survival of stevia. Greenhouse and field trials were conducted to assess the effect of multiple fungicides on plant growth, root associated microorganisms, and overwintering survival. In greenhouse trials in the absence of soilborne pathogens, no differences in plant height, shoot weight, or root system ratings were observed among treatments. In two field trials screening seven combination fungicides and one biological control, significant increases in overwintering survival of plants treated with QoI fungicides were observed. Plants were dug from the fields monthly to destructively sample root crowns and species of Fusarium, Ceratobasidium, Pythium, and Macrophomina phaseolina were consistently isolated. Abundant microsclerotia from M. phaseolina were observed on stems of plants that did not survive winter. In seven sample months at each of the two field trials, non-fungicide treated plants had the lowest root weights. This study is one of the first to evaluate interactions between fungicide use and root associated fungi in a perennial crop and provides a framework to further investigate the role of root-associated fungi in overwintering survival.}, journal={Crop Protection}, publisher={Elsevier BV}, author={Koehler, A.M. and Shew, H.D.}, year={2019}, month={Jun}, pages={13–20} }
 @article{koehler_larkin_rogers_carbone_cubeta_shew_2019, title={Identification and characterization of Septoria steviae as the causal agent of Septoria leaf spot disease of stevia in North Carolina}, volume={111}, ISSN={0027-5514 1557-2536}, url={http://dx.doi.org/10.1080/00275514.2019.1584503}, DOI={10.1080/00275514.2019.1584503}, abstractNote={ABSTRACT Stevia (Stevia rebaudiana) is an emerging perennial crop in the southeastern United States. A Septoria leaf spot disease of stevia was first identified on field plantings in Japan in 1978. The pathogen was named Septoria steviae based on a morphological characterization. In 2015, a species of Septoria with morphological characters of S. steviae was isolated from field and greenhouse-grown stevia plants with leaf spot symptoms in North Carolina. In this study, 12 isolates obtained from diseased stevia plants in 2015 and 2016 were characterized and compared with reference strains of S. steviae. Comparisons were based on conidial and pycnidial morphology and multilocus sequence analysis of actin (ACT), β-tubulin (BT), calmodulin (CAL), nuc rDNA internal transcribed spacers (ITS1-5.8S-ITS2 = ITS), nuc rDNA 28S subunit (28S), RNA polymerase II second largest subunit (RPB2), and translation elongation factor-1α (TEF1). Measurements of conidia and pycnidia from symptomatic field leaves and 12 pure cultures grown on nutrient medium were consistent with those previously reported for ex-type strains of S. steviae. North Carolina strains formed a well-supported monophyletic group with ex-type strains of S. steviae. This study represents the first genetic characterization of S. steviae in the United States and provides an experimental framework to elucidate the genetic diversity and disease ecology of field populations of S. steviae.}, number={3}, journal={Mycologia}, publisher={Informa UK Limited}, author={Koehler, Alyssa M. and Larkin, Maximo T. and Rogers, Layne W. and Carbone, Ignazio and Cubeta, Marc A. and Shew, H. David}, year={2019}, month={Apr}, pages={456–465} }
 @article{koehler_shew_2018, title={Field efficacy and baseline sensitivity of Septoria steviae to fungicides used for managing Septoria leaf spot of stevia}, volume={109}, ISSN={["1873-6904"]}, DOI={10.1016/j.cropro.2018.03.006}, abstractNote={Koehler, A.M. and Shew, H.D. 2018. Field efficacy and baseline sensitivity of Septoria steviae to fungicides used for managing Septoria leaf spot of stevia. Crop Protection 106:000–000. Stevia (Stevia rebaudiana) is an herbaceous perennial emerging as a new crop in the southeastern US. Septoria leaf spot caused by Septoria steviae is present across all production areas in North Carolina, causing leaf lesions that expand and result in total defoliation when left unmanaged. Fungicide efficacy trials for management of Septoria leaf spot were conducted over 2 years at two field sites. Seven fungicides, single or combination products, reduced disease severity and increased yield compared to non-fungicide treated controls. Azoxystrobin, chlorothalonil, fluopyram, fluxapyroxad, pyraclostrobin, and tebuconazole were screened using an in vitro assay to establish sensitivity profiles for 10 isolates of S. steviae that had received 0 or 1 year of fungicide exposure. All S. steviae isolates were sensitive to all fungicides evaluated. Successful management of Septoria leaf spot is critical for long term establishment of stevia as a crop in the southeast US.}, journal={CROP PROTECTION}, author={Koehler, A. M. and Shew, H. D.}, year={2018}, month={Jul}, pages={95–101} }
 @article{koehler_shew_2017, title={Enhanced Overwintering Survival of Stevia by Qol Fungicides Used for Management of Sclerotium rolfsii}, volume={101}, ISSN={["1943-7692"]}, DOI={10.1094/pdis-02-17-0277-re}, abstractNote={ Stevia (Stevia rebaudiana) is a herbaceous perennial under evaluation as a new crop in the southeastern United States. Stem rot caused by Sclerotium rolfsii is common in stevia plantings in North Carolina, with symptoms including wilting, root and stem necrosis, and plant death. Fungicide efficacy trials for management of S. rolfsii were conducted over 2 years. Fungicides evaluated included azoxystrobin, flutolanil, and tebuconazole applied at three timings. Azoxystrobin applied to transplants 1 week prior to planting had the lowest area under the disease progress curve values across all trials. Fungicide plots were also used to evaluate overwintering of stevia. End-of-season stand counts were compared with spring emergence counts to quantify overwintering survival. In spring 2015, plots treated with azoxystrobin in 2014 had greater overwintering survival (78%) than other fungicide treatments (38%) and the control (38%). Similar results were obtained at two locations in spring 2016 in plots treated with azoxystrobin or pyraclostrobin in 2015. Successful overwintering of stevia directly impacts the profitability of second- and third-year harvests and enhances the likelihood of long-term establishment of stevia as a viable crop. Future studies will be directed at elucidating the mechanism of the enhanced overwintering survival of plants treated with quinone outside inhibitor fungicides. }, number={8}, journal={PLANT DISEASE}, author={Koehler, A. M. and Shew, H. D.}, year={2017}, month={Aug}, pages={1417–1421} }
 @article{koehler_shew_2018, title={First Report of Charcoal Rot of Stevia Caused by Macrophomina phaseolina in North Carolina}, volume={102}, ISSN={["1943-7692"]}, DOI={10.1094/pdis-05-17-0693-pdn}, abstractNote={HomePlant DiseaseVol. 102, No. 1First Report of Charcoal Rot of Stevia Caused by Macrophomina phaseolina in North Carolina PreviousNext DISEASE NOTES OPENOpen Access licenseFirst Report of Charcoal Rot of Stevia Caused by Macrophomina phaseolina in North CarolinaA. M. Koehler and H. D. ShewA. M. Koehler†Corresponding author: A. M. Koehler; E-mail: E-mail Address: [email protected]Search for more papers by this author and H. D. ShewSearch for more papers by this authorAffiliationsAuthors and Affiliations A. M. Koehler † H. D. Shew , Department of Entomology and Plant Pathology, North Carolina State University, Raleigh, NC 27695. Published Online:24 Oct 2017https://doi.org/10.1094/PDIS-05-17-0693-PDNAboutSections ToolsAdd to favoritesDownload CitationsTrack Citations ShareShare onFacebookTwitterLinked InRedditEmailWechat Stevia (Stevia rebaudiana [Bertoni] Bertoni) is an emerging perennial crop in the United States. Small black root lesions were first observed on overwintering crowns dug in March 2016 from a field planting of stevia in Rocky Mount, NC. Similar root lesions were observed on first year plants in Kinston and Rocky Mount, NC, throughout the 2016 growing season in fields with a history of soybean and corn rotations. Isolations from lesions were made on potato dextrose agar amended with 50 µg/ml of streptomycin sulfate and penicillin G. Isolations were incubated at room temperature (22 to 25°C) for 1 week and observed for colony morphology. Gray-black colonies that became dark with age were isolated into pure culture. Hyphae were septate with barrel-shaped cells, and numerous 50 to 150 µm microsclerotia resembling those produced by Macrophomina (Kaur et al. 2012) were present. Pathogen identification was confirmed by sequencing the internal spacer (ITS) region of ribosomal DNA. DNA was extracted from mycelia with the Qiagen DNEasy Plant Mini Kit (Qiagen, Valencia, CA) and subjected to a PCR using universal primers ITS 4,5. Isolates were identified as Macrophomina phaseolina (100% sequence identity with GenBank accession no. KF951775.1). Koch’s postulates were confirmed on 10-week-old stevia plants, cv. G3, grown in 8-cm-diameter pots in the greenhouse. Ten 6.75-mm agar plugs were buried 1 cm deep approximately 2 cm from the base of the plant in each of six replicate sterile pots. Plants were observed over a 6-week period for symptom development, at which time root necrosis similar to field symptoms was evident on all inoculated plants. Noninoculated plants did not develop symptoms. M. phaseolina was reisolated from necrotic inoculated roots and confirmed by morphology and sequencing. To our knowledge, this is the first report of M. phaseolina causing charcoal rot of stevia in North America. Hilal and Baiuomy (2000) reported M. phaseolina as the causal agent of charcoal rot on stevia in Egypt. At this time, no fungicides are registered for stevia. Since stevia is often grown as a perennial rotation crop, and M. phaseolina was present year-round in roots of stevia, it will be important to determine if this pathogen becomes more damaging over a three year production cycle and reaches levels that may have negative impacts on rotation crops.References:Hilal, A., and Baiuomy, M. 2000. Egypt. J. Agric. Res. 78:1435. Google ScholarKaur, S., et al. 2012. Crit. Rev. Microbiol. 38:136. https://doi.org/10.3109/1040841X.2011.640977 Crossref, ISI, Google ScholarDetailsFiguresLiterature CitedRelated Vol. 102, No. 1 January 2018SubscribeISSN:0191-2917e-ISSN:1943-7692 Metrics Article History Issue Date: 20 Dec 2017Published: 24 Oct 2017First Look: 29 Aug 2017Accepted: 26 Aug 2017 Pages: 241-241 Information© 2018 The American Phytopathological SocietyCited byFirst Report of Macrophomina euphorbiicola Causing Charcoal Rot of Stevia in ParaguayAndres D. Sanabria-Velazquez, Alberto Cubilla, Maria Eugenia Flores-Giubi, Javier E. Barua, Cristina Romero-Rodríguez, Guillermo A. Enciso-Maldonado, Lindsey D. Thiessen, and H. David Shew10 January 2023 | Plant Disease, Vol. 107, No. 1Charcoal rot of Stevia rebaudiana caused by Macrophomina phaseolina in Brazil10 August 2022 | Journal of Plant Pathology, Vol. 104, No. 4Different histone deacetylase inhibitors reduce growth, virulence as well as changes in the morphology of the fungus Macrophomina phaseolina (Tassi) Goid28 February 2022 | World Journal of Microbiology and Biotechnology, Vol. 38, No. 4Macrophomina phaseolina (charcoal rot of bean/tobacco)CABI Compendium, Vol. CABI CompendiumTrichoderma asperellum as a preventive and curative agent to control Fusarium wilt in Stevia rebaudianaBiological Control, Vol. 155Nondestructive Sampling to Monitor Macrophomina phaseolina Root Colonization in Overwintering SteviaLayne W. Rogers and Alyssa M. Koehler24 March 2021 | Plant Health Progress, Vol. 22, No. 2Full Issue PDF9 June 2022 | Plant Health Progress, Vol. 22, No. 2One stop shop III: taxonomic update with molecular phylogeny for important phytopathogenic genera: 51–75 (2019)17 September 2019 | Fungal Diversity, Vol. 98, No. 1Effects of fungicide applications on root-infecting microorganisms and overwintering survival of perennial steviaCrop Protection, Vol. 120}, number={1}, journal={PLANT DISEASE}, author={Koehler, A. M. and Shew, H. D.}, year={2018}, month={Jan}, pages={241–241} }
 @article{koehler_lookabaugh_shew_shew_2017, title={First report of pythium root rot of stevia caused by Pythium myriotylum, P. irregulare, and P. aplzanidermatum in North Carolina}, volume={101}, number={7}, journal={Plant Disease}, author={Koehler, A. M. and Lookabaugh, E. C. and Shew, B. B. and Shew, H. D.}, year={2017}, pages={1331–1332} }
 @article{koehler_shew_2017, title={Seasonal dynamics and fungicide sensitivity of organisms causing brown patch of tall fescue in North Carolina}, volume={109}, ISSN={["1557-2536"]}, DOI={10.1080/00275514.2017.1377587}, abstractNote={ABSTRACT Brown patch, caused by multiple species of Rhizoctonia and Rhizoctonia-like fungi, is the most severe summer disease of tall fescue in home lawns across the southeastern United States. Home lawns were surveyed in central North Carolina from 2013 to 2015 to determine the organisms present during typical epidemics of brown patch in tall fescue. Isolates of Rhizoctonia and Rhizoctonia-like fungi were obtained by sampling 147 locations in July 2013 and May and July 2014. In addition, 11 sites were sampled once a week for 12 consecutive weeks from late May to the end of July 2015. All isolates were identified to species and anastomosis group with nuc rDNA internal transcribed spacer (ITS) sequence analysis. Isolations from brown patch lesions in May 2014 predominately yielded Ceratobasidium cereale (77% of the organisms recovered), whereas the organisms recovered in July 2013 and 2014 were R. solani AG 2-2-IIIB (44%), R. solani AG 1-IB (37%), and R. zeae (14%). In 2015, Ceratobasidium cereale was isolated from all 11 locations in May but was replaced by Rhizoctonia species in June and July. Sensitivity of the May 2014 isolates to multiple concentrations of the fungicides azoxystrobin, flutolanil, fluxapyroxad, and propiconazole was compared with sensitivity of isolates collected in 2003, to determine whether multiple years of exposure to fungicides applied for brown patch control had altered fungicide sensitivity. Historical isolates of R. solani, which had never been exposed to fungicide applications for brown patch control, were also included for comparison. Mean EC50 values (concentration of fungicide needed to inhibit mycelial growth by 50%) varied across fungicides and species, but no resistance was observed, and there was no apparent shift in sensitivity over the years. An additional 94 isolates from 2015 were screened against azoxystrobin, flutolanil, fluxapyroxad, and propiconazole, and fungicide insensitivity was not observed.}, number={4}, journal={MYCOLOGIA}, author={Koehler, Alyssa M. and Shew, H. David}, year={2017}, pages={667–675} }
 @article{koehler_shew_2014, title={First Report of Stem Rot of Stevia Caused by Sclerotinia sclerotiorum in North Carolina}, volume={98}, ISSN={["1943-7692"]}, DOI={10.1094/pdis-03-14-0307-pdn}, abstractNote={ Stevia (Stevia rebaundiana Bertoni) is an emerging perennial crop in the United States. The crop is grown for 3 to 5 years with two harvests per growing season. Stevia contains numerous glycosides that are used as a natural noncaloric sweetener, and in 2008 was approved by the USDA as a sugar substitute. In commercial plantings of second-year stevia in North Carolina, diseased plants were observed in April and May of 2013. Diseased plants were observed in several counties in the state in fields that had been planted primarily in a corn-soybean rotation prior to stevia planting. Symptoms included wilting, chlorotic leaves, necrotic leaves at the base of the stem, bleached stem lesions, and dead plants. Symptomatic plants often also had tufts of white hyphae present on stems and large, irregularly shaped 2- to 8-mm black sclerotia frequently were present on the base of the stem. Isolations from infected stem tissue were made on potato dextrose agar amended with 50 μg/ml of streptomycin sulfate and penicillin G. Based on hyphal and sclerotial characteristics, isolates were tentatively identified as Sclerotinia sclerotiorum (Lib.) de Bary (4). Koch's postulates were confirmed on 10-week-old Stevia plants cv. G3 grown in the greenhouse in 10-cm-diameter pots containing a sterile 1:1:1 sand, loam, media mix. Oat grains infested with one isolate obtained from diseased field plants served as the inoculum. Oats were sterilized on three consecutive days, inoculated with colonized agar plugs of S. sclerotiorum, and then incubated at room temperature until they were thoroughly colonized. Three infested oat grains were buried 1 cm deep approximately 2 cm from the base of the plant in each of the six test pots and plants were observed over a 3-week period for symptoms. Symptoms developed on all plants within 5 days of inoculation. Leaves began to wilt, then turned chlorotic and necrotic, with stem lesions and sclerotia present at the base of the plant. Isolations were taken from infected stem tissue and pure cultures were prepared for molecular identification. Uninoculated control plants did not develop symptoms. Pathogen identification was confirmed using universal primers ITS 4,5 and β-tubulin (2,3). Mycelium from the cultured greenhouse stem isolations were grown in potato dextrose broth. Mycelium samples were aspirated and lyophilized prior to DNA extraction. Extracted DNA was amplified through PCR with ITS and β-tubulin primers and sent for sequencing. Sequences were aligned using CLC Workbench. Sequences from ITS45 had 100% identity to S. sclerotiorum GenBank Accession No. KF859933.1, confirming S. sclerotiorum as the causal organism. The β-tubulin sequence was compared against the Broad Institute S. sclerotiorum whole genome shotgun sequence and was confirmed to have 100% identity to the beta tubulin chain (5). This is the first report of S. sclerotiorum on stevia in the United States. Chang et al. (2) reported a stem rot of stevia in Canada and confirmed S. sclerotiorum as the causal organism.  References: (1) K. Chang et al. Plant Dis. 81:311, 1997. (2) J. Freeman et al. Eur. J. Plant Pathol. 108:877, 2002. (3) N. L. Glass and G. C. Donaldson. Appl. Environ. Microbiol. 61:1323, 1995. (4) J. E. M. Mordue and P. Holliday. CMI No. 513, 1976. (5) Sclerotinia sclerotiorum Sequencing Project, Broad Institute of Harvard and MIT. Online: http://www.broadinstitute.org/ , accessed July 16, 2014. }, number={10}, journal={PLANT DISEASE}, author={Koehler, A. and Shew, H.}, year={2014}, month={Oct}, pages={1433–1433} }